The impact of morphology on the cellulose beads’ potential application was investigated in the context of drug loading and release. The results show that the fibrous morphology showed a better loading capacity than the globular analogue due to its higher surface area and pore volume.Cellulose beads were successfully prepared from waste denim using a dissolution-regeneration approach with ionic liquids as the dissolving solvent. Cellulose beads with different morphologies were achieved by altering the dissolving and coagulating solvents. The morphological differences were quantified by N The negative environmental impacts of the textile industry have been in the spotlight in recent years due to the increasing public awareness around sustainability and climate change. One of the pressing issues faced by the textile industry is the increasing amount of textile waste ,2. GlobaOne such advanced product is cellulose beads, also known as cellulose particles or microspheres. It is a high-performance material with a wide range of applications ,11,12. CIn our previous study on cellulose aerogels , it was Waste denim fabrics are made of 100% cotton. Sodium hydroxide (NaOH) (anhydrous pellets) was purchased from Sigma-Aldrich . Two ionic liquids (ILs), 1-butyl-3-methylimidazolium chloride (BmimCl) and 1-butyl-3-methylimidazolium acetate (BmimAc) were purchased from IOLITEC, Heilbronn, Germany. Both ILs were used as received.The two selected drugs, theophylline (Thp) and lidocaine hydrochloride monohydrate (LiHCl), were purchased from Sigma Aldrich. Distilled water was used as the solvent to prepare drug solutions.As illustrated in The dissolution of denim in both ILs was conducted at 100 °C with magnetic stirring. The completeness of dissolution was verified by means of polarised microscopy when no crystalline structure could be observed. The denim concentration was fixed at 4 wt.% in this study.The denim-IL solution was transferred into a 3-mL syringe and was extruded at a constant rate . While the solution was being extruded from syringe nozzle, two methods were adopted for producing beads. For the cellulose-BmimAc solution, a blade was used to shear off the solution to form a bead. For cellulose-BmimCl, the solution was too viscous to be sheared off; therefore, scissors were used to cut the solution into spheres. The spherical shaped droplets were then dropped directly into a coagulation bath. Distilled water and ethanol were chosen as coagulation baths. The bath was changed every 1 h in the first 4 h and then immersed in water for 24 h. When ethanol was used as the coagulation bath, the obtained beads were washed with distilled water to exchange ethanol with water before drug loading. The rheological measurements of the cellulose–IL solutions were conducted on an ARES-G2 Rheometer  equipped with a Peltier temperature control system and a 40 mm parallel geometry. The gap was set at 50 mm for all samples. Oscillation frequency sweep measurements of the solution were conducted at 25 °C in the frequency range of 0.01 to 100 rad/s and the strain was set at 0.1%. Complex viscosity, storage modulus, and loss modulus were recorded.2 drying was used to remove anti-solvents without influencing the solid content of cellulose beads so that the characterisations of the inner pore structure could be determined. All of the wet beads were washed with excessive ethanol to replace water and were then immersed in ethanol for 12 h before drying. The drying was performed on a Leica critical point drier . The porosity of the supercritical-dried beads was analysed by N2 physisorption measurement on Quantachrome Autosorb iQ3 . Samples were degassed at 80 °C for 10 h before being subjected to the 80-point physisorption measurements. The surface area was obtained by Brunauer–Emmett–Teller (BET) theory analysis on relative pressure 0.05–0.25, and the pore size distribution was analysed from the desorption curve based on the Barrett-Joyner-Halenda (BJH) theory with the exclusion of points with relative pressure below 0.3 [The diameters of the fully coagulated beads were measured using a Vernier calliper and were controlled within 2.0 ± 0.1 µm. Thirty beads were measured for each type of beads. Supercritical COelow 0.3 ,32. ScanLidocaine hydrochloride monohydrate (LiHCl) and theophylline (Thp) were selected as the representative substances, which are commonly employed for evaluating the drug delivery efficiency of different porous materials ,33,34. LFor each drug, a concentration measurement was carried out to correlate the absorption value with the solution concentration using the Cary 300 UV-Vis spectrometer . Four concentrations of drug solutions were prepared with 0.1 N HCl and the absorption values were recorded. The standard concentration curves of LiHCl and Thp are shown in The as-prepared wet beads were immersed in drug solutions for 24 h. The loaded beads were then removed from the drug solutions and dried in air overnight. The amount of incorporated drugs was obtained on the basis of the weights of dried beads before and after loading the drugs. Each batch involved 15 beads, and all the experiments were repeated three times. The loading capacity is calculated as follows:The dried loaded beads (15–25 beads in each batch) were immersed in 250-mL 0.1 N HCl buffer solution and constantly stirred at a fixed stirring rate of 100 rpm at 37.0 ± 0.5 °C. The release protocol is designed according to the USP paddle method with minor adjustments. The stirring paddle was maintained at the same position for each test. Four millilitres of the solution was taken for UV-Vis measurement at fixed five-minute time intervals for the first 20 min and then every 10 min from 20 to 80 min. The samples were taken at the same position each time to avoid deviations. All the experiments were performed in triplicate. The amount of the drug released was calculated using the obtained concentration–absorption curve. In the end, the drug release of each sample was plotted according to the percentage of the initial amounts versus time. The release efficiency is calculated as follows:The swelling capacity of the beads was measured using a gravimetric method . The dryClwater has a cross-section that comprises a dense fibrous network at the nanoscale , a change from globular to fibrous network was observed, similar to that of BmimClwater ; when ethanol was used as the coagulating solvent for the cellulose-BmimAc sample  is the same (34.3 nm), which is significantly larger than the globular morphology (13.9 nm). This is likely due to the considerably higher diffusion rates of the solvent and anti-solvent in the case of BmimAcwater. The cumulative pore volumes of the fibrous beads are consequently larger than that of globular beads with the larger average pore size. Between the two fibrous beads, BmimAcethanol has a higher cumulative pore volume than BmimClwater, thus indicating a higher porosity. As shown in Acwater have a weight of 0.44 ± 0.04 mg, which is higher than that of BmimAcethanol (0.26 ± 0.01 mg) and BmimClwater (0.35 ± 0.05 mg). This indicates a higher solid content in BmimAcwater than the other two beads and thus a lower porosity. This result is consistent with the different surface areas of the beads. Overall, the porosities of beads prepared from waste denim here are comparable with the reported cellulose beads and aerogels in various shapes, the pore sizes of which are mostly mesopores (2–50 nm), resulting in an surface area of 200–500 cm3/g [00 cm3/g ,39.Acethanol shows the highest loading capacity for both drugs, incorporating 43% and 10% for LiHCl and Thp, respectively  and loading capacity has been previously found in investigations of various drug delivery systems [The loading of the drug was achieved by immersing beads in the drug solution for 24 h. The amount of incorporated drug in the bead was determined by the weight difference of the dry bead before and after the drug loading. LiHCl and Thp were chosen as model drugs . Because systems ,40.Acwater shows the lowest initial release for both drugs, which is likely due to a smaller average pore diameter. The smaller pore diameter could lead to more capillary condensation that enables more compact storage in the pores. When the beads are immersed in the suitable media, the absorbed drug molecules diffuse out in an ordered manner. The molecules that are stored inside the nanopores have to queue up, thus resulting in a sustained release behaviour. The release curves for BmimAcethanol and BmimClwater are similar, as they both have a fibrous morphology. The swelling capacity of the beads is an important characteristic that influences the diffusion of the absorbed substrate. Acwater, has the lowest swelling capacity. This is consistent with the lower release profile, while both fibrous bead types have similar swelling capacities.The loaded beads were then placed in 0.1 N HCl at 37.0 ± 0.5 °C, which mimics the stomach environment, to test their in vitro release profile over an 80-min period, as suggested by Yildir et al. . The relThree types of cellulose beads were prepared from waste denim with distinct morphologies and porosities by varying the IL used for dissolution and the coagulating solvent. By characterising the porous structure and examining the drug delivery profiles of three types of beads, it can be concluded that the loading capacities of the cellulose beads were positively correlated with the nanoporous characteristics of the beads (mainly the surface area and cumulative pore volume). Cellulose beads produced from BmimCl in water performed the best among the varieties, with 43% of their weight loaded with LiHCl. The subsequent drug release studies showed that 70%–90% of the loaded drug was released in the first five minutes across an 80-min measurement period. Surface functionalisation can be applied to cellulose to further improve the loading and release profiles of cellulose beads or achieve certain controlled release according to the desired performance for specific drugs and targeted releasing locations in the human body.


Pentalonia nigronervosa) and carried long distances through the movement of infected plant materials. The banana aphids harboring banana bunchy top virus (BBTV) present in banana producing regions are the sole vector and the most efficient method of transmitting the virus to the healthy plants. Controlling the spread of BBTD has been very challenging since no known banana germplasm is immune to BBTV. The disease can be managed with the use of virus‐free planting material and roguing. However, once BBTD is established in the field, it is very difficult to eradicate or manage it. Therefore, a more sustainable way of controlling the disease is developing host plant resistance against the virus and the vector. Biotechnological strategies via RNA interference (RNAi) could be used to target the banana aphid as well as BBTV to reduce virus‐associated yield losses of banana and plantain, which feed over 500 million people around the world. This review discusses the status of BBTD and perspectives on effective RNAi technologies for controlling BBTV and the vector, banana aphid, transmitting the virus as sustainable management of the disease.Banana bunchy top disease (BBTD) is one of the world's most destructive viral diseases of banana and plantain, causing up to 100% yield loss in severe cases. The disease is vectored by banana aphids ( banana bunchy top virus__(BBTV) to reduce virus‐passociated yield losses of banana and plantain, which feed over 500 million people around the world. This review discusses the status of BBTD and perspectives on effective RNAi technologies for controlling BBTV and the vector, banana aphid, transmitting the virus as management of the disease.Banana bunchy top disease (BBTD) is one of the world's most destructive viral disease of banana and plantain causing up to 100% yield loss in severe cases. Biotechnological strategies via RNAi could be used to target banana aphid as well as__ BBTD was first reported in the 1880s in Fiji  has been identified as one of the main biotic and economically important constraints to banana and plantain production worldwide Dale, . It is ri Magee,  and has i Magee, . In Afrii Magee, . In 2018i Magee, . This isi Magee, . Fruit pMusa germplasm. Biotechnological approaches could offer a pathway to improving and/or activating the defense mechanism of plants to diseases. The use of advanced biotechnological approaches such as host plant‐mediated RNA interference (RNAi) against BBTV and banana aphids has potential for the management of BBTD. The RNAi strategy has been demonstrated for the control of several viruses and insect vectors (Tables Plant resistance is the most appropriate method to control BBTD. There is no identified immunity to BBTV in the s Tables  and 2. T2Banana bunchy top virus belongs to the genus Babuvirus and the family Nanoviridae. It is a complex circular single‐stranded DNA (ssDNA) virus that multiplies within the phloem tissue of a host plant  responsible for initiating viral DNA replication, DNA‐S encodes the coat protein (CP), DNA‐C encodes the cell‐cycle link protein (Clink), DNA‐M encodes the movement protein (MP), and DNA‐N encodes the nuclear shuttle protein (NSP), while the function of DNA‐U3 is unknown  found on various host plants such as ginger, tomatoes, taro, Xanthosoma spp., cardamom, Heliconia spp., calla, and costus  are the Musa species and Ensete , genome prevented transmission of the virus by an aphid , which may be used to create improved banana/plantain varieties. However, there is no known germplasm with immunity or high levels of resistance to BBTD. Although there has been variation in susceptibility among some cultivars in the banana germplasm  or activating degradation of sequence‐specific RNA  and Tomato yellow leaf curl China virus (TYLCCNV), AC2/AL2 protein of Begomoviruses, C2/L2 protein of Curtoviruses, betasatellites (βC1) of TYLCCNV, cysteine‐rich protein (CRP) and triple gene block protein 1 (TGBp1) of Potato virusM (PVM), helper component proteinase (HC‐Pro) of potyviruses such as Turnip mosaic virus (TuMV), Tobacco etch virus (TEV), 2b of Cucumber mosaic virus (CMV), P38 of Turnip crinkle virus (TCV), P19 of Cymbidium ring spot virus (CRSV), viral genome‐linked protein (VPg) of potyviruses, P1 helper component proteinase (P1/HC‐Pro) of potyviruses, P6 of Cauliflower mosaic virus (CaMV), CP and P6 of Olive mild moaic virus (OMMV), and MP of babuviruses, dianthoviruses and citriviruses are some of the examples of VSRs that have been identified to inhibit RNA silencing , inhibiting the key steps of RNAi system to neutralize the RNAi‐based antiviral defense of the host plant. Several proteins such as V2 protein of the The CP and MP of BBTV encoded by DNA‐S and DNA‐M have been identified as suppressors of RNAi  or partial coding sequence of Rep gene together with its 5′ partial upstream regulatory region of the BBTV. The transgenic plants showed resistance against BBTV up to 6 months postinoculation with viruliferous aphids. The prospect of RNAi relies on a comprehensive understanding of the multipartitism of BBTV and its synergistic interaction with its host and downstream biochemical machinery, which would elucidate how to improve the specificity of RNAi for virus transmission control.Nevertheless, RNAi has proven to be a useful approach in preventing the expression of viruses in plants Table . It has 7Banana bunchy top virus is very difficult to manage, and not much success has been achieved toward developing host plant resistance through breeding or transgenic approach. Therefore, controlling virus transmission by aphids represents a new alternative for the management of aphid‐borne BBTD. One option could be to block the aphid‐mediated transmission of the virus by interfering with aphid–virus interactions to inhibit virus acquisition by aphids. The efficiency of virus acquisition is one critical parameter in determining aphid transmission efficiency. The aphid transmission can be blocked by impairing the virus acquisition at the gut epithelial cell barrier via direct interference with aphid–virus protein interactions. The receptors and/or virus‐binding protein(s) present on the gut and accessory salivary gland promote the transmission specificity  genes encoding a nucleoside diphosphate kinase were significantly expressed in wingless than in winged morphs in A. gossypii and identified to play a significant role in the development and differentiation of insects  gene in the green peach aphid, and transgenic plants were resistant to aphids  transmission by aphids. The study indicated that a 5‐day spray of BCMV‐CP dsRNA on the plants successfully protected Nicotiana bentamiana and Vigna unguiculata plants from infection when exposed to viruliferous aphids (Worrall et al., Efficacy of dsRNA has also been linked to the mode of uptake of dsRNA into insect pest which could be through injection, ingestion, or a recently modified topical application of dsRNA using nanoparticle technology to form a stable and sustained dsRNA delivery to protect plants against insect pests and has been applied to green peach aphid (Basnet & Kamble, 8P. nigronervosa and BBTV. Therefore, it is imminent that a holistic approach required for control of the spread of BBTD through the use of RNAi‐based technology is feasible.The advances in biotechnological approaches to inhibit the destructive potentials of pests and diseases open up avenues for crop improvement. BBTD is becoming a global pandemic in banana and plantain producing regions, if not controlled. The attributed huge production losses intimate a significant threat to banana and plantain production, which is a major crop that sustains smallholder farmers and contributes to the annual income of several countries in the world. Managing the spread of vector‐borne plant diseases is a sustainable way to increase crop production. The control approach will depend on the intended outcome, which could either suppress banana aphid's population or inhibit its ability to transmit BBTV, causing BBTD. RNAi is a promising strategy, which can control both the virus and insect vector transmitting the virus. The delivery of RNAi into the host plants can be transgenic or nontransformative alternatives that are topically applied dsRNA Figure . The effThe authors declare no conflict of interest.


The objective is to report recent data on the infection and detection of Zika virus in infertile couples and to discuss the need to make disease surveillance compulsory in this population in order to decrease the burden on the healthcare system and expedite treatment onset.We collected and analyzed the results of Zika virus infection screening tests of infertile couples in a private clinic in the low-incidence region of Curitiba - Brazil.Among the 1189 serologies performed, 98.5% were negative for Zika virus, 0.75% were positive, and 0.75% were inconclusive. The twenty-one reverse transcription polymerase chain reaction tests performed for confirmation of infection were negative.Zika virus infection screening for asymptomatic patients may lead to delayed fertility treatment initiation in addition to excessive expenses for the patients. Based on our results, we challenge the validity of mandatory screening, especially in low-incidence regions. Zika virus is a flavivirus initially identified only in mosquitoes in several African countries. In 1953, it was isolated in three Nigerian patients, representing the first record of the pathogen in humans. From this episode until 2007, when the first outbreak occurred in Micronesia, only 13 cases had been reported. Thereafter, other outbreaks occurred in the Pacific Islands. In March 2015, Zika virus was first identified in the Americas when an outbreak of exanthematous disease occurred in the state of Bahia and from there it spread throughout the Brazilian territory . The most common symptoms identified during the outbreak in Micronesia were pruritic macular or papular skin rash, fever, arthritis or arthralgia, non-purulent conjunctivitis, myalgia, and headache  tests that detect the viral RNA (ribonucleic acid) or by detection of specific IgM and IgG antibodies in the serum. Detection of the virus by RT-PCR is possible only during the symptomatic period, when the virus is present in the bloodstream, which reduces the window of opportunity for detection. Immunological tests can detect signs of infection for longer periods, because IgM becomes positive approximately 1 week after infection and persists for several months  (The Food and Drug Administration (FDA) published a resolution with guidelines for Zika virus screening to reduce virus transmission in tissue and cell donors. Patients diagnosed with Zika virus infection, those having traveled to affected areas within the last 6 months, or women having had sexual intercourse with a man who had traveled to an affected area within the last 6 months should be considered ineligible for donation .Given the alarming number of patients infected with Zika virus and the evidence for vertical transmission, the National Agency for Health Surveillance (ANVISA) published recommendations for germ cell and tissue banks. The new guidelines recommend that patients scheduled to undergo treatments involving manipulation of gametes (whether their own or from donors) should be serologically tested for Zika IgM antibodies. The test samples need to be collected no more than 5 days prior to the oocyte collection procedures. Only patients with negative test results can be referred for collection. In cases of inconclusive or positive results, the tests need to be repeated after 30 days for confirmation; alternatively, molecular tests for infection markers can be performed at any time .Considering the low incidence in the South region, the objective of this communication was to report recent data on the infection and detection of Zika virus in infertile couples and to discuss the need to make disease surveillance compulsory in this population in order to decrease the burden on the healthcare system and expedite treatment onset.The local ethics committee approved the study. Due to the retrospective nature of the study, patients were not required to give consent.in vitro fertilization or intrauterine insemination) at a private clinic in the city of Curitiba  between April 2016 and November 2019. We included all the results obtained during this period. Patients from geographical regions other than the South were excluded. We calculated the absolute frequency of positive and negative cases. In addition, we calculated the number of cases confirmed by PCR among those with positive IgM antibodies. The results were obtained from a spreadsheet designed to preserve patient confidentiality.This was a descriptive, retrospective study involving the statistical analysis of screening tests results (serology and RT-PCR) for Zika virus in couples undergoing a cycle of assisted reproduction . Among them, 1,171 (98.5%) yielded negative results, 9 (0.75%) were positive, and 9 (0.75%) were inconclusive. In all 21 RT-PCR tests performed, the results were all negative.The 9 positive serological tests belonged to 7 different patients. Four of them also had RT-PCR tests done (with negative results), one was serologically re-tested and had an inconclusive result, and two patients didn’t return after the result.The nine serologies with inconclusive results also belonged to different patients. Four of them (one mentioned above) did not have follow-up tests after their inconclusive results; four had the tests repeated after 25 days and obtained negative results, and one had an RT-PCR test done after 29 days with a negative result as well.Zika virus spread throughout Brazil until February 2017, and more than 70,000 cases were reported. However, in 2016, the incidences of the infection in the southern regions were drastically lower  .In addition, in 2016, more than 10,000 children with developmental changes possibly associated to Zika virus infection were reported throughout Brazil. However, the southern regions reported the least number of cases (only 2.2%) . ResearcIn addition to the financial issues imposed by Zika virus screening, the tests in asymptomatic patients may significantly delay the start of infertility treatments because screening should be repeated within a minimum of 25 days to begin a new cycle of treatment. In some cases, the delay in starting treatment can lead to worse results and greater anxiety for the couple undergoing assisted reproduction.The study has limitations related to its population and sample characteristics. We collected the data retrospectively, and the examination results were obtained from patients assisted in a private clinic. Considering that infection with Zika virus is correlated to important social factors, the analyzed population may not faithfully reflect the general population.Our results indicated a low rate of positive tests, which is consistent with the results of other studies. Therefore, our results make us challenge the validity of Zika virus screening for asymptomatic patients, particularly in low-incidence regions. Compulsory screening may lead to increased anxiety about the fertility treatment with possible poorer outcomes due to delayed therapy initiation and increased financial burden.


Peas are an important legume for human and animal consumption and are also being used as green manure or intermediate crops to sustain and improve soil condition. Pea production faces constraints from fungal, bacterial, and viral diseases. We investigated the virome of German pea crops over the course of three successive seasons in different regions of pea production to gain an overview of the existing viruses. Pools from 540 plants, randomly selected from symptomatic and asymptomatic peas, and non-crop plants surrounding the pea fields were used for ribosomal RNA-depleted total RNA extraction followed by high-throughput sequencing (HTS) and RT-PCR confirmation. Thirty-five different viruses were detected in addition to nine associated nucleic acids. From these viruses, 25 are classified as either new viruses, novel strains or viruses that have not been reported previously from Germany. Pea enation mosaic virus 1 and 2 were the most prevalent viruses detected in the pea crops, followed by pea necrotic yellow dwarf virus (PNYDV) and turnip yellows virus which was also found also in the surrounding non-legume weeds. Moreover, a new emaravirus was detected in symptomatic peas in one region for two successive seasons. Most of the identified viruses are known to be aphid transmissible. The results revealed a high virodiversity in the German pea fields that poses new challenges to diagnosticians, researchers, risk assessors and policy makers, as the impact of the new findings are currently unknown. German pea virome. Pisum sativum L.) are popular vegetables in Germany. The production of green peas increased from 4,444 ha in 2010 to 5,488 ha in 2018  has enabled the identification of many new viruses from domesticated and wild plants , d. HTS In this study, using HTS we explored the spatio-temporal changes of the pea virome in six German regions with different production foci, including frozen produce, protein pea seed production, seed production and breeding over a period of three seasons. We also investigated alternative virus reservoirs of legume and non-legume weeds associated with these production sites. To our knowledge, this is the first metagenomics study of crop plants incorporating spatio-temporal developments.Six regions in Germany were chosen for sampling and included Salzlandkreis-1: pea seed production, Salzlandkreis-2: trial site and heritage pea collection, Münster: trial site pea breeding, Kreis Stormarn: protein pea production, Landkreis Rostock: trial site for green manure mixtures, and Landkreis Meißen: green pea frozen production for human consumption.A typical production field was chosen and based on long-term experience, samples were selected randomly over three successive seasons  and four separate pools comprising material from all SP (60 plants), aSP (60 plants), sL (30 plants), or snL (30 plants), respectively, from all regions . Fresh tTM Plant Kit for RNA-Seq (Invitrogen). cDNA was synthesized using ProtoScript II Reverse Transcriptase (NEB) and random octanucleotide primers (8N), followed by second strand synthesis using a NEBNext Ultra II Non-Directional RNA Second Strand Synthesis Module (NEB). The libraries were prepared from the double-stranded cDNA using Nextera XT Library Prep Kit (Illumina). The HT sequencing was performed on an Illumina MiSeq platform (301 × 2).Total RNA was extracted using innuPREP Plant RNA Kit  from three subsamples of 100 mg from each pool to ensure sufficient total RNA yield for further processing thus allowing detection of low titre viruses. The extracted subsets were mixed and used for ribodepletion. Ribosomal RNA (rRNA) was depleted using a RiboMinusDe novo assembly was performed and the resulting contigs were compared against a local database of viruses and viroids sequences downloaded from NCBI using Blastn and Blastx  (downloaded 13 August 2018). The generated consensus sequences were based on the highest quality threshold. Primers for virus validation were designed using a modified version of Primer3 (2.3.7) tool in Geneious Prime . The reads were quality trimmed and normalized. us Prime . Pairwisus Prime . Neighbous Prime . The phyFor virus identity confirmation, total RNA was re-extracted from each pool as described above, followed by RT-PCR with the primers listed in Statistical analysis was performed using scripts written on R (version 3.5.3) . The virSymptoms observed on peas in the “symptomatic” pea pool included plant dwarfing, stunting of top leaves, translucent leaf spotting and leaf enations, leaf yellowing, leaf mosaic and mottling, leaf rolling and pod distortion . In 2016Luteoviridae was represented by seven species members, followed by Secoviridae with six members, and Potyviridae with five members. Over the survey, the widest virus diversity was recorded in the SP pools, with sixteen different virus species present. The sL pool contained 12 different virus species, the snL pools included 11 virus species, and the aSP recorded seven virus species  was the most abundant virus, with a score of 5, followed by white clover cryptic virus 2 (WCCV2) and soybean dwarf virus (SbDV) with a score of 2 . All theTuYV was the most abundant virus from the study and was detected not only in pea (SP and aSP) but also in snL pools over the three seasons. Nucleotide identity (nt%) showed 95–99.2% to published TuYV sequences . Two assOver the observation period, PEMV1 was detected almost all the time in all regions in SP and aSP pools with the exceptions of region 5 in 2017 and region 4 in 2018, where neither PEMV1 nor PEMV2 could be detected. The nucleotide (nt) sequences of PEMV1 isolates showed highest identities (96.1–98.9%) to MK948533 . In genePNYDV was detected in SP pools with nt identities between 99.2 and 99.9% with PNYDV sequences available on NCBI . Six PNYPSbMV was detected only in two regions, Salzlandkreis-2 and Kreis Stormarn, but was present over all three seasons in SP and aSP pools. PSbMV isolates had 96.2–99.7% nt identity to D10930, a Danish isolate. Neighbor joining tree based on the polyprotein aa sequences of the potyvirid isolates detected in German pea fields over three seasons are shown in Emaravirus including fig mosaic virus (FMV), pigeonpea sterility mosaic virus 2 (PPSMV2) and rose rosette virus (RRV). Members of the genus Emaravirus  have segmented, linear, single-stranded, negative-sense RNA genomes (A new emaravirus was identified in SP pools from of Landkreis Meißen in 2017 and 2018. The virus showed high similarity to other members of genus  genomes . Their g genomes . The aa  genomes . TherefoNarnaviridae) were detected in the SP and aSP pools in 2017. An isolate of Sclerotinia sclerotiorum mitovirus 4 (SsMV4) was identified from the SP pool. This isolate shared 96.3% aa identity to SsMV4 from New Zealand (AGC24233). The three further mitoviruses identified are provisionally called pea associated mitovirus 1, 2 and 3 . The PaMV1-CDS shared closest identity of 68.8% nt to Erysiphe necator mitovirus 3 , identified from the grape powdery mildew fungus Erysiphe necator (Schwein.) [syn. Uncinula necator (Schw.)], recently described in the United States  shared closest aa identity to LSV1 with 67.3% while its CP region showed closest aa identity to LSV1 with 31.6%. Neighbor joining tree based on the Pro-Pol aa sequences of the secovirids detected in German pea fields over three seasons are shown in Trivirinae) in the snL of Münster 2017, sharing 74.4% identity with carrot chordovirus 1. Finally, in 2018, a partial waikavirus sequence was detected from the snL pool of Kreis Stormarn and appears to be related to bellflower vein chlorosis virus. A phylogenetic tree based on the alignments of the replicase aa sequences of the betaflexivirids is shown in In Münster 2017, a new putative member of the family Vitivirus; subfamily: Trivirinae, family: Betaflexiviridae), was identified in snL of Münster 2017.In addition to the viruses described above, new virus strains were detected including red clover enamovirus 1 (RCEV1), red clover umbravirus (RCUV), red clover nepovirus A (RCNVA), red clover carlavirus 1 (RCCV1) and Wuhan insect virus 21 (WHIV21) were identified in the sL, and divergent isolates of cherry leaf roll virus (CLRV) and carrot torradovirus 1 (CaTV1) were found in the snL. Also, the complete coding sequence of an isolate of Heracleum latent virus (HLV)  was identified in the sL samples. Its RdRp aa sequence shows 87.1% identity to the Czech strain (MG596229). Another isolate was detected in Landkreis Meißen in 2017, with 87.6% nt aa identity to each other and 95.8% to the Czech strain. The viruses grouped together within the us clade . A new sus genus .Carlavirus; subfamily: Quinvirinae; family: Betaflexiviridae), was identified in one location (Kreis Stormarn) in 2018 only. The partial RdRp sequence shared 85.3% aa identity with RCCV1 (MG596238 and MG596239) from the Czech Republic. The CDS of an isolate of Heracleum latent virus (HLV) , was identified the snL of Münster 2017. Based on the CP sequence, this isolate shared 90.9% identity to HTV (NC_039087) on the nt level and 96.4% identity based on the aa sequence. Moreover, the RdRp shared 58.4% nt identity to grapevine virus B  and 58.9% aa identity.A divergent strain of red clover carlavirus 1 (RCCV1) , was detected in Landkreis Rostock 2017. This new strain was identified with Pro-Pol aa identity of 96.5% to MG253828 and CP aa identity of 83.2% to MG253829. A divergent cherry leaf roll virus (CLRV) (genus: Nepovirus) was identified in Salzlandkreis-1 in 2018. The virus shared closest identity with CLRV isolates from New Zealand, where RNA1 has 82.4% nt identity to CLRV isolates KC937022 and RNA2 shared 80% to KC937029. The aa sequence of the Pro-Pol region is 97% identical to KC937022 while the CP region is 89.8% to KC937029. A new strain of carrot torradovirus 1 (CaTV1) with similarity to CaTV1 strain celery from Germany (MK063924 and MK063925) with 95.9% aa identity to the Pro-Pol region and 95.4% to the CP region.In addition, a new strain of RCNVA  was detected in sL of Salzlandkreis-1 2018.The spatial and temporal compositions of the virome, in the different regions over a period of three growing seasons, show similarities as well as fundamental differences. In pea crops, PEMV1, PEMV2, and PNYDV were the viruses that were found in all regions but not in every season . PEMV1, PSbMV was detected in all seasons in Salzlandkreis-2 and Kreis Stormarn. BLRV was detected only in Salzlandkreis-2 in 2016 and 2017 but it could not be found in 2018. PaEV was also detected for two seasons, in 2017 and 2018, only in Landkreis Meißen.TuYV was detected in all three seasons in Salzlandkreis-1, Münster and Landkreis Meißen; however, after finding it present in Kreis Stormarn in 2016, but it could not be detected in 2017 and 2018. In 2017 TuYV was also not detected in Salzlandkreis-2. TuYV was never detected in the Landkreis Rostock region .Other detected viruses included BYMV, detected only in Salzlandkreis-1 and Salzlandkreis-2 in 2018. ClYVV was found in Salzlandkreis-2 in 2018 and Münster in 2017. RCEV 1 and RCUV were detected only in Landkreis Meißen in 2017; however, both viruses were detected in the sL of Münster in 2016. SbDV was detected in SP in Münster 2016 and in the sL of Kreis Stormarn in 2016 and 2018. BLRV and PaEV were detected on SP only in Salzlandkreis-2 and Landkreis Meißen, respectively.In summary, from the spatial distribution of the 12 pea viruses (excluding the mitoviruses), we observed that the highest virus occurrence was detected in Salzlandkreis-2 (17 occurrences over the three seasons) followed by Landkreis Meißen with 14 virus occurrences. A total of 13 pea virus occurrences were reported from Salzlandkreis-1, 12 for Münster, nine for Kreis Stormarn and the lowest number of occurrences was for Landkreis Rostock (only five pea viruses). The temporal virus occurrences appeared to be relatively stable over the successive seasons with the highest being 2016 with 27 occurrences, then 22 for 2017 and 21 for 2018 .This is the first HTS-based study to describe the pea virome in Germany and to our knowledge, elsewhere in the world. In addition to the focus on viruses infecting pea crops, we also explored spatio-temporal aspects of virus infection across six different production regions over a period of 3 years. We also explored potential virus reservoirs by investigating legume and non-legume hosts in and around the crops. Our study is distinct from many metagenomics studies in that we explored our subject over three seasons in six regions. Other studies have focused on either just one crop plant, one production area, one season or a combination thereof. We believe that this study demonstrates the importance of incorporating spatio-temporal elements into metagenomics studies to help us to draw a more complete picture of all the viruses present and their impacts on the host crop as we would have missed many virus detections if we had focused on either just one growing season or just one region .To explore the pea virome dispassionately, we used ribosomal RNA-depleted total RNA for our sequencing approach . This meUsing RT-PCR, we were able to confirm the presence of pea viruses that are commonly known to be present in Germany including PEMV1, PEMV2, TuYV, PNYDV, BLRV, SbDV, PSbMV, and CMV. PEMV1 and PEMV2 are the most common viruses found in German pea crops . PEMV diBrassicaceae (vice versa (data not shown). It is therefore very likely that rapeseed crops, other members of the Brassicaceae family as well as a large number of common weeds and wild species are alternative hosts for pea-infecting poleroviruses such as TuYV  Fuss. At this stage, their biological relevance is still unclear  in the Czech Republic . Relationships between luteovirids and umbraviruses are important, for example, in the relationship between PEMV1 and PEMV2 where one virus provides cell-to-cell movement within hosts and the other virus’ coat protein is required for genome packaging and vector transmission , which indicates that more information is required to determine if surrounding perennial legumes are a virus reservoir for these viruses. Mixed infections of both viruses were confirmed in red clover . RRV causes extreme damage to roses, leading to plant death within a short period of time (Cajanus cajan L. (Fabaceae): pigeonpea sterility mosaic disease (SMD), caused by pigeonpea sterility mosaic emaravirus 1, and pigeonpea sterility mosaic emaravirus 2 (Aceria cajani Chann.)  . One can Chann.) . The numAphanomyces, and/or fusarium root rot diseases in peas (Sclerotinia sclerotiorum (Lib.) de Bary, a widespread plant pathogenic fungus, which causes white mold disease especially in peas, lentils and beans and many other hosts (Sclerotinia sclerotiorum mitovirus 2 (SsMV2) and Sclerotinia sclerotiorum mitovirus 3 (SsMV3), reduced the in vitro growth and virulence of S. sclerotiorum on cabbage, common bean, oilseed rape and tomato  in the Czech Republic  and a novel ilarvirus, sLaIV. In 2016, a survey using antibodies developed for detection of red clover vein mosaic virus (RCVMV)-like carlaviruses suggested the presence of a carlavirus in several pea samples but the exact virus species was not determined . KoloniuRepublic . It was Republic .In the pools of non-legume (snL) plants, we detected viruses previously described in Germany. These are CaTV1, CLRV, and TuYV. TuYV was often detected and poses a threat to other commercial crop plants including rapeseed and sugar beet. CaTV1 was recently detected in celery plants, exhibiting chlorotic ringspots, mosaic and strong yellowing symptoms . VirusesIn summary, our study represents the first comprehensive virome study of the pea crop covering pea production regions, temporal effects and alternative hosts. We believe that our strategy using HTS was successful. We not only detected well established pea viruses such as PEMV and PNYDV, but also discovered viruses not previously reported from Germany, as well as a number of new viruses. Furthermore, we also detected viruses in samples that appeared asymptomatic indicating that HTS is able to detect early stage or low titer infections and/or asymptomatic infections. The sequence data that we generated will also improve our knowledge of virus taxonomy, ecology, epidemiology and host plant resistance. The primer that we developed can be applied by diagnosticiancs to check for the presence of the novel viruses and virus-associated nucleic acids. It also demonstrates the challenges of metagenomic HTS studies in the framework of laboratory and bioinformatics, result interpretation, biological significance, pest risk analyses and data sharing as we also found viral reads that could not be attributed to “real” virus findings .While we acknowledge the strength of HTS in identifying known and unknown viruses of crops, our pooling strategy has disadvantages. Firstly, we cannot obtain detailed information on the viruses infecting a single plant without back-testing each specimen in the pool. Secondly, it was not always possible to recover the full-length viral sequence using this method. Thirdly, pooling does not allow us to link individual plants symptoms with the viruses detected.Luteoviridae, Nanoviridae, Potyviridae, and Tombusviridae families. In addition, 25 new viruses associated with pea, non-crop legumes and non-legume plants were found, some unexpected and yet unexplained. More work is needed to reveal the importance and context of these new host/virus associations.In conclusion, our method of using rRNA-depleted total RNA extracts from pooled plant tissue in combination with HTS, bioinformatic analysis and molecular confirmation has increased the speed and breadth of virus detection in one crop species in Germany, over three seasons. This method enabled the detection of a range of viruses regardless of their genome type. After sequencing our samples, we identified thirty-five viruses, for which we obtained many nearly full genomes . As expeWe found PEMV1 and PEMV2 were the dominant virus species in pea, which is consistent with what has been observed in the past. The most abundant virus was TuYV, as its sequences were not only recovered from pea pools but particularly from non-leguminous alternative host plants, which is consistent with its wide host range. A new emaravirus was detected in peas over two of the survey seasons, but its method of transmission and impact are still unknown and need further investigations. Other viruses were also detected in pea or alternative plants for the first time in Germany and their impacts have yet to be determined. Interestingly, most of the viruses detected in this survey were aphid-transmitted, thus emphasizing the continued importance of aphid management to reduce virus-spread. We also found viruses with little similarity with known species and suggest they could be categorized as unclassified. Many of these viruses have yet to be included in standard monitoring programs of pea diseases and therefore the abundance and impact of these viruses on pea and other legume crops are unknown.We believe the data from this study provides a comprehensive and improved overview of viruses present in German pea fields, and demonstrates the usefulness of HTS and metagenomics. For the newly detected viruses, further work is needed to determine the complete host range of these viruses, their effect on hosts and their likely vectors. It is also necessary to further investigate different locations and environments to increase our understanding of the diversity of these new viruses, not only for pea crops but also for other legumes, on a global scale. Our study shows that there are many more viral players in the field(s) than known from the past. Further studies are required to address the risks that these new viruses might pose to peas or other crops. Targeted surveys could assess the distribution and abundance of these new players and the risk they might pose to crop plants.https://www.ncbi.nlm.nih.gov/genbank/, MN314973, MN399680–MN399748, MN412725–MN412751, and MN497793–MN497846.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: YG, JF, AB, RM, and HZ conceived and designed the experiments. YG, JF, AB, and HZ performed the sampling. YG performed the experiments, analyzed the data, and wrote the draft of the manuscript. KH and JH provided technical assistance. RM and HZ acquired the funding. All authors read and approved the final version.JF, AB, and RM were employed by The New Zealand Institute for Plant and Food Research Limited, Auckland, New Zealand. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Concerns have recently been raised about reported incidents of intestinal obstruction following ingestion of Superabsorbent polymer (SAP) beads. Texas Poison Centers reported 110 cases of superabsorbent polymer bead ingestions between 2011 and 2016 . Furthermore, cases of related auditory complications following the placement of SAP beads into the external auditory canal have also been reported. Here, we report the first case of significant airway damage secondary to the ingestion of a SAP bead (Orbeez), which was aspirated and then overlooked. Further, we hypothesized that the capability of the bead to expand in size once exposed to water from the respiratory mucous may contribute to airway damage.A 3-year-old boy presented to our hospital with persistent cough and recurrent hospitalizations to the general ward and intensive care unit. The boy was diagnosed with focal lung bronchiectasis in the left lower lobe, which occurred after the patient aspirated an Orbeez bead before a year. The bead was removed using flexible bronchoscopy and a retrieval basket.Orbeez beads are commonly ingested by young children resulting in gastrointestinal obstruction. The beads can easily be aspirated by children and overlooked by their caregivers and physicians for long periods of time due to their small size. The bead can cause significant airway damage after multiplying in size when coming into contact with respiratory mucus which consists of 95% water. Foreign body aspiration is relatively common public health problem. Accounting for 7% of accidental deaths in children under 4 years of age , 2. DespThe longer the FB remains in the bronchial tree the greater the chances are of it becoming dislodged from the bronchus and impacting the larynx, with risk being higher in inorganic FBs .Superabsorbent polymer (SAP) bead capable of increasing its original size up to 200 times once placed in water . It has In this study, we report the first case of significant focal lung damage as a result of an Orbeez bead ingestion which expanded in size after coming into contact with lower airway secretions. Thus, this study necessitates spreading public awareness of the potential danger of SAP beads.non-consanguineous, and there was no history of respiratory infections or symptoms in the family. Upon examination of the patient’s immune system, test results showed normal immunoglobulin levels and normal lymphocyte markers. A computed tomography (CT) scan of the chest  from the regional hospital in Al-Ahsa region in the eastern province of Saudi Arabia. The patients experienced recurrent chest infections associated with a persistent wet cough which resulted in four hospitalizations over the last year. The child was born uneventfully after stable pregnancy and had no remarkable background history in the first 2 years of his life apart from infrequent mild upper respiratory tract infections without symptoms, suggestive of a lower respiratory tract infection. Shortly after his second birthday, the boy had the first onset of lower respiratory tract infections and presented to the local hospital with fever, tachypnea, and respiratory distress, significant enough to require ICU admission. He responded partially to the initial regimen with oxygen therapy, systemic antibiotics, and systemic steroid and nebulized bronchodilators during the hospitalization. His cough persisted after he was discharged from the hospital and he soon suffered from additional attacks during which his symptoms worsened. His symptoms included respiratory distress, an aggravated cough, and hypoxia, and required frequent emergency visits and a total of 4 hospitalizations over the course of 1 year. The patient’s parents are Clinical examination performed at our facility demonstrated a normal oxygen saturation on room air. There was a decrease in air entry in the left lower zone with no adventitious sounds. Initial chest x-ray Fig.  showed aAn attempt was made to remove the foreign body using a retrieval basket and a flexible scope. Unfortunately, the foreign body was squashed and fragmented into smaller pieces, which required the removal of each piece using the retrieval basket. Eventually, all fragments were removed successfully. We observed that granulation tissue was causing a significant obstruction of the left lower lobe posterior segment airway, to the extent that we could not pass the scope through it. After reconstructing the pieces of the foreign body, the patient’s parent indicated that it appeared much larger than its original size. The fragmented pieces were reviewed by a pathologist who confirmed the inorganic nature of the foreign body. At an evaluation after 6 months following the removal of the foreign body the child’s symptoms were minimal, with no additional hospitalizations recorded. His chest X-ray demonstrated a persistent bronchiectasis in left lower lobe with interval improvement in the previously noted consolidation Fig. .Fig. 4CSAP beads are small round objects made of inorganic material. They are an inexpensive and widely available children’s toy with the appearance of candy .Concerns have been raised after various reports of intestinal obstruction following ingestion of these beads , 10.Our study described a case in which the immediate symptoms were unremarkable. Nevertheless, after coming into contact with respiratory mucus which consists of 95% water, the beads increased in size, causing damage to the lungs . If swalAge restriction and parent observation are not an effective way to prevent these SAP ingestion from occurring, as nearly half of the reported incidents occurred in school-aged children, with 30% occurring while the children were at school .Thus far, studies have reported that toys such as SAP beads to cause harm to the intestines, the external auditory canal, and as in our case, the respiratory system. We suspect that if stuck in the larynx and enlarged after absorbing water, SAP beads may result in a catastrophic sequelae.For the above mentioned reasons, we propose recalling all brands of SAP beads from markets. In the meantime, we suggest that every incidence of complications following the ingestion of SAP beads be reported.


We evaluated the initial and follow-up treatment costs of different post-restorations in a practice-based German healthcare setting.n = 68), glass-fiber , or cast post-and-core buildups  had been used to retain crowns or bridge anchors. Preformed metal and glass-fiber had also been used to retain directly built up post-retained composite restorations . Material and treatment costs for the initial post-restorations as well as restorative, endodontic, or surgical re-treatments were estimated from a public-payer-perspective in Germany. Associations between total and annualized total costs and covariates were assessed using generalized linear modeling. The study was registered in the German Clinical Trials Register (DRKS-ID: DRKS00012938).A total of 139 incisors, canines, or premolars received post-restorations placed by eight general dental practitioners in Germany, and were followed over a mean ± SD 7.1 ± 4.5 years. Preformed metal .MC showed highest initial treatment costs (557.23 Euro), but the least re-treatments , while PC showed lowest initial costs (203.52 Euro) but the most re-treatments . Costs for MP/GF post-crowns were initially similarly costly (496.47/496.87 Euro), and both also showed similar re-treatments (35%/36%). The overall annual failure rate was 5.2% . Including costs for the resulting re-treatments, mean total costs were 591.66 Euro (MC), 548.31 Euro (MP), 526.37 Euro (GF), and 361.81 Euro (PC). Annualized total costs were 171.36 Euro (MC), 141.75 Euro (MP), 146.12 Euro (GF), and 135.65 Euro (PC). Total and annualized total costs were highest for MC, with PC being the significantly less costly option  contains supplementary material, which is available to authorized users. For endodontically treated teeth with extensive loss of coronal hard tissue, post placement is often performed to allow a subsequent core buildup. A number of previous investigations have assessed success and survival of post-restorations, showing limited annual failure rates of such restorations, at 0–2% per year.  Most of For post-restorations, direct preformed metal (MP) or indirect cast metal (MC) posts, or non-metal glass-fiber (GF) posts can be employed. Theoretically, different post materials may come with different risks of root fracture , 3 and dA recent health economic study, employing a modeling approach based on data mainly from randomized trials, found only limited cost-effectiveness differences between different post-restorations.  Over a pTo resolve this, long-term, practice-based, prospective studies are needed, which allow to determine effectiveness and costs over relevant horizons, with sufficient depth of recording to allow economic analyses without the need to rely on models. The findings in these realistic settings are not only relevant for clinicians but also for payers and patients. A recent study on post-restorations from a practice-based network in Germany, for example, found much higher annual failure rates than most previous studies from other settings.  Based onThis prospective, practice-based cohort study did not need an ethical approval according to the local review board of the ethical committee of the Medical Chamber of Lower Saxony. Patients’ consent was not required given this being a retrospective evaluation of non-personal data. The study has been registered in the German Clinical Trials Register (DRKS00012938). Note, however, that the present analysis on long-term costs was not planned a priori, but is an auxiliary, exploratory analysis. Reporting of this study follows the STROBE  and CHEEIn this prospective, practice-based cohort study, involving eight general practices from a German dental practice–based research network (Arbeitskreis Zahnärztliche Therapie), patients receiving minimum one post-restoration were included. The cohort has been described in more detail before.  Briefly,For the present study, the following data were assessed from electronic case report forms: Initial and re-interventions, with materials for the post, the core, and the coronal restoration, as well as possible restorative re-treatments , endodontic re-interventions , or extractions . For teeth experiencing extractions, no data on prosthetic replacements for the teeth were provided, though, which may lead to costs being under-estimated. If repeated re-treatments were required, this was recorded, too. In addition, confounding variables like the name of the private practice and the dentist, the patient’s age and gender, the number of missing teeth, the tooth type, and if it was the last tooth in the dental arch or not, were recorded. Teeth and restorations were re-assessed via clinical and intraoral radiographic examinations in individual intervals. Examinations were performed by the dentist who placed the post-restoration, and while criteria for failure and treatment decisions had been discussed among the dentists beforehand, examiners were not calibrated; intra- or inter-examiner reliability data were not available.The health economic analyses were performed in the context of German healthcare. Our outcome parameter was costs. The study’s horizon was determined by the follow-up of patients, with teeth either being extracted, or follow-up being concluded (censored). As teeth might have been retained after follow-up ended (censoring), our study did not aim to assess cost-effectiveness, but only costs per year of follow-up.Costs were calculated in Euro 2018, assuming all patients have been provided with the post-restoration in 2018. This eases interpretation for today’s reader but may not capture the true costs borne by patients or the health insurances at the time. Future costs  were discounted at 3% per annum . DiscounA public-payer’s perspective was chosen. Dental treatments in Germany are largely reimbursed by the statutory (public) insurance, with the majority of German patients (87%) being enrollees.  For thesTreatment costs within the statutory insurance are estimated using the statutory fee item catalog, Bewertungsmaßstab (BEMA). Laboratory costs were covered by the Bundeseinheitliches Leistungsverzeichnis (BEL-II). Details on items and cost estimation (using item-points being transformed into Euro) are provided within the appendix Tables . We did Initial treatment costs included those for the initial clinical and radiographic examination, post placement, core buildup, a temporary post and a temporary crown if needed, and a crown being placed, assuming this to be a full metal crown for reasons of standardization. In case no crown, but a composite restoration was placed, costs for a four-surfaced incrementally placed restoration, including additional efforts for moisture control and matrix application, were applied. Moreover, material and laboratory costs for the models, dye and resin materials, metals if needed, and technical manufacturing of the restorations, were estimated. Re-treatment costs included those for assessment, radiographs if needed, re-cementation, crown or composite restoration renewal, post renewal using the same or a completely new coronal restoration, apicectomy, and extraction, with local anesthesia if needed.Costs were calculated using a spreadsheet . Costs were discounted, and total and annualized costs covering initial and re-treatments estimated. We additionally assessed follow-up costs only. Association of total and annualized total costs with patient- and tooth-level parameters was evaluated via generalized linear mixed models , accounting for clustering and also accounting for follow-up. A reduced set of covariates was employed in a sensitivity analysis. Statistical analysis was performed using SPSS 22 . Visualization was done using the Python plotting library Matplotlib. n = 68; (2) GF plus crowns, n = 28; (3) metal or glass-fiber posts in combination with directly built up, post-retained composite restorations ; and (4) MC, n = 23.A total of 139 post-restorations were included and followed over a mean ± SD (min.–max.) 7.1 ± 4.5 (0.2–13.7) years. The following post materials and the coronal restorations had been used: (1) MP plus crowns, MC showed the highest initial treatment costs (557.23 Euro), but the least re-treatments , while PC showed lowest costs (203.52 Euro) but most re-treatments . Costs for MP/GF were initially similar (496.47/496.87 Euro), and both also showed similar re-treatments . The mean annual failure (re-treatment) rate was 5.2% . During follow-up, annual costs (SD) for MC, MP, GF, and PC were 7.12 (21.92), 10.58 (19.65), 10.42 (21.97), and 34.91 (83.34) Euro, respectively.The results of initial and follow-up costs are total costs. These were the highest for MC, and lowest for PC; this was also applicable when total costs were annualized. Total follow-up costs, in turn, were highest for PC, and lowest for MC, also when follow-up costs were annualized Table . Total cp < 0.001). Total costs differed significantly between post-restorations . Similarly, annualized total costs were significantly lower in PC than in MC (p < 0.001), if accounting for age, dental arch, tooth type, missing teeth, treating dentists, tooth position, and follow-up period as confounders. Annual costs also increased significantly with follow-up . After a mean of 7 years, the initially least costly treatment, PC, nevertheless remained significantly less costly than the initially most expensive option, MC, despite requiring more re-treatments.Our primary outcome (costs) was the result of both the initial costs and the need for re-treatments, i.e., the risk of failure. For post-restorations, as discussed, annual failure rates of around 2% have been recorded,  while soThis is the first practice-based study assessing long-term costs of post-restorations. Based on our findings, both initial but also re-treatment costs for post-restorations are significant. With annualized costs of 135–171 Euro in mean, retaining teeth via post placement may, overall, have only limited cost-effectiveness compared with alternative strategies, like tooth replacement. Note that these costs are also much higher than those estimated using a modeling approach, mainly built on data from randomized trials (with lower risks of failure), finding annualized cost of around 27 Euro. This may be partially explained by the longer horizon of the modeling study, where the high initial costs were distributed over a mean follow-up period of 26–28 years (decreasing them to around 20 Euro per tooth and year). The re-treatment costs in both studies were at around 7–10 Euro per year, i.e., similar, but it should be highlighted that in the modeling study, these included costs for prosthetic replacement of extracted post-teeth; this was not the case in the present study. Overall, it seems that post-restorations come with high initial and considerable re-treatment costs, being a rather costly and high-maintenance treatment strategy.We further associated total and annualized total costs with a range of confounding variables. As discussed, post and also coronal restoration type were significantly associated with costs, especially if annualized costs were assessed . Patients’ age, gender, number of missing teeth, and tooth type or location were not significantly associated with costs. Data on risks of restorative failure along genders are ambiguous, , 23 so wThis study is subject to a number of limitations. First, with a sample size of 139 teeth, it is most likely underpowered to detect association between costs and some covariates. Larger sample sizes or more balanced class distribution  may increase the power. Second, the setting, a practice-based network in Germany, and the study design, a prospective observational study, are prone to a number of biases. Dentists joining such networks may not be representative for all dentists in Germany, and neither may the care provided nor the patients treated. The dentists did not allocate treatments at random; it is conceivable that certain treatments  may have been placed based on indications , so causal associations may not apply. The same dentist who provided the treatment also assessed its success during follow-up. Again, bias is likely here. Overall, the applicability of our findings may be limited when transferred to other settings, and caution is needed when assessing the found associations. The limited generalizability also applies to other types of post-crown types, for example double crowns retaining removal dentures, where costs (and longevity) will differ. Third, restorations in the present study were pragmatically classified according to both the post material and the coronal restoration. Notably, we merged glass-fiber and preformed metal posts into one group if they served to retain a direct composite restoration (PC), mainly as the PC group was of limited sample size. Stratifying it further would have increased uncertainty and decreased statistical power. Last, our cost estimation was built on fee items from the statutory insurance in Germany. These have been found to reflect the true direct medical costs to some degree, and studies using these yielded comparable results with studies from other settings, with other methods of cost estimation. –28 NeverWithin the limitations and generalizability of this study, both initial and follow-up costs for post-restorations were considerable. Saving costs initially may, at least partially, be offset by more complications long term. Comparative controlled studies should assess the cost-effectiveness of retaining teeth using post-restorations versus alternative treatment strategies. Exploring between-dentist variation in costs seems worthwhile, too.ESM 1(DOCX 32 kb).


Endemic to West Africa and South America, mammalian arenaviruses can cross the species barrier from their natural rodent hosts to humans, resulting in illnesses ranging from mild flu-like syndromes to severe and fatal haemorrhagic zoonoses. The increased frequency of outbreaks and associated high fatality rates of the most prevalent arenavirus, Lassa, in West African countries, highlights the significant risk to public health and to the socio-economic development of affected countries. The devastating impact of these viruses is further exacerbated by the lack of approved vaccines and effective treatments. Differential immune responses to arenavirus infections that can lead to either clearance or rapid, widespread and uncontrolled viral dissemination are modulated by the arenavirus multifunctional proteins, NP and Z. These two proteins control the antiviral response to infection by targeting multiple cellular pathways; and thus, represent attractive targets for antiviral development to counteract infection. The interplay between the host immune responses and viral replication is a key determinant of virus pathogenicity and disease outcome. In this review, we examine the current understanding of host immune defenses against arenavirus infections and summarise the host protein interactions of NP and Z and the mechanisms that govern immune evasion strategies. RNA viruses, despite the limited size of their genomes, pose serious challenges to global public health ,2,3. A mPublic health concerns have heightened due to the increased incidence of arenavirus epidemics in endemic countries; this is worsened by the lack of vaccines and treatments available. The ongoing, devastating outbreaks of Lassa virus (LASV), the most prevalent arenavirus endemic to Western Africa, highlight the importance of understanding the nature and intricacy of key interactions that occur during emerging RNA virus infections ,7,8,9. SArenaviridae family is a large group of diverse, enveloped, negative-sense RNA viruses. Members of this family are classified into four distinct genera: Antennavirus, Mammarenavirus, Hartmanivirus, and Reptarenavirus, based on their natural reservoir hosts—fish, rodents, and reptiles for both Hartmanivirus and Reptarenavirus, respectively AP- and/or PPxY-type) motifs, known as late domains, that drive virus particle release through the recruitment of ESCRT proteins that result in the final cellular membrane scission stage required for budding ,174,175.Like NP, the Z protein is also able to regulate the host cell interferon system ,184. SeqCurrently, very limited strategies exist to treat arenavirus infection and rely on early treatment with ribavirin and its analogues, that have very little to no impact on fatality rates ,188. In Arenaviruses utilise host cellular machinery to negotiate host defenses. The remarkably multifunctional viral proteins NP and Z have evolved distinct and synergistic mechanisms to evade the antiviral state induced upon virus infection ,155,184.


The current review offers the first documented evidence on cost estimates of diabetes in Malaysia.Diabetes causes significant disabilities, reduced quality of life and mortality that imposes huge economic burden on societies and governments worldwide. Malaysia suffers a high diabetes burden in Asia, but the magnitude of healthcare expenditures documented to aid national health policy decision-making is limited. This systematic review aimed to document the economic burden of diabetes in Malaysia, and identify the factors associated with cost burden and the methods used to evaluate costs. Studies conducted between 2000 and 2019 were retrieved using three international databases  and one local database (MyCite), as well as manual searches. Peer reviewed research articles in English and Malay on economic evaluations of adult type 2 diabetes conducted in Malaysia were included. The review was registered with PROSPERO (CRD42020151857), reported according to PRISMA and used a quality checklist adapted for cost of illness studies. Data were extracted using a data extraction sheet that included study characteristics, total costs, different costing methods and a scoring system to assess the quality of studies reviewed. The review identified twelve eligible studies that conducted cost evaluations of type 2 diabetes in Malaysia. Variation exists in the costs and methods used in these studies. For direct costs, four studies evaluated costs related to complications and drugs, and two studies were related to outpatient and inpatient costs each. Indirect and intangible costs were estimated in one study. Four studies estimated capital and recurrent costs. The estimated total annual cost of diabetes in Malaysia was approximately USD 600 million. Age, type of hospitals or health provider, length of inpatient stay and frequency of outpatient visits were significantly associated with costs. The most frequent epidemiological approach employed was prevalence-based ( Type 2 diabetes is a metabolic disease characterized by progressive loss of adequate β-cell insulin secretion, frequently because of insulin resistance . The prePeople with diabetes are predisposed to macrovascular complications such as cardiovascular, cerebrovascular, and peripheral vascular diseases, as well as microvascular complications like retinopathies, nephropathies and neuropathies ,8,9. TheWhile the impact of diabetes to society is notable, the economic costs borne by healthcare systems and governments are hugely substantial. The burden of diabetes amplifies significant direct and indirect consequences to patients. While the direct costs deal with healthcare resource consumption such as medications, outpatient and inpatient costs, the indirect costs largely encompass monetary losses caused by disabilities and deaths, travel expenses, nutrition costs, productivity and income loss . FinanciIt is crucial to understand resource utilization of the allocated budget to manage the diabetes burden and its complications. Succinctly, Malaysia has commenced using pharmacoeconomic evidences for regulatory assessments of new drugs . These tEconomic assessments require identification and measurement of costs related to the illness, treatment or program under evaluation. Commonly identified costing components within the healthcare system are categorized into direct, indirect, intangible, capital, recurrent and per diem costs ,16,19,20Direct costs are correlated with resources used to manage an illness. Direct costs are regarded as the primary cost of healthcare programs and often include expenditure for medical care or treatment of the disease. Directs costs are typically subdivided into direct medical costs  and direct nonmedical costs .Indirect costs are secondary costs associated with paid or unpaid productive activities. Such costs can emerge when treatment is confined within the hospital or home, causing the expenses to be incurred by patients or their families due to productivity loss as a result of significant morbidity, disability or mortality.Intangible costs are nonquantifiable costs. These include social, emotional and human costs that do not convert to money, such as pain, suffering or loss of quality of life.Capital costs are costs that evaluate buildings, land, vehicles and equipment that require special consideration. Such costs arise at a single point of time, but the assets are used over a period of time.Recurrent costs include staff emoluments that are involved in the management of the disease, overhead costs such as operation and maintenance of the building, equipment or vehicles. It also includes consumables, drugs, and investigation costs, such as laboratory, radiological, and ECG tests.Per diem costs refer to per bed-day costs for patients admitted into the hospital, which include administrative, food and kitchen service costs. It measures resource consumption for each patient but excludes costs directly related to medical care, such as drugs or special consumable items.A comprehensive systematic search of the literature regarding economic burden and cost evaluation of type 2 diabetes mellitus in Malaysia was conducted between September and October 2019 following the Preferred Reporting Items for Systematic Review and Meta-Analyses (PRISMA) guidelines . This syMajor English databases that include PubMed, Scopus and EMBASE, and the Malaysian only database, Malaysian Citation Index (MyCite), were explored to identify relevant studies reporting cost evaluation of diabetes and its complications among adults in Malaysia between 1 January 2000 and 31 August 2019. In addition, a manual search of the reference lists in the articles included for the review was used to identify further eligible articles. Only original research articles in English and Malay from peer-reviewed publications were included.Search terms and their combinations are presented in Articles were included if they provided original research findings related to costs  of type 2 diabetes mellitus and its complications in Malaysia following the Population, Intervention, Comparison, Outcome, Study (PICOS) type approach for systematic reviews. Population considered were people with type 2 diabetes mellitus in Malaysia; the contexts of interest were hospitals, primary healthcare clinics and home settings; direct, indirect, intangible, capital and recurrent costs from the healthcare (provider) and individuals’ perspective comprised the outcomes; and relevant study designs were observational , randomized controlled trials (RCTs) and modeling studies.Articles that reported on the economic burden of diabetes using both quantitative and qualitative methods were included to extract data related to costs. An extraction table using an Excel spreadsheet was developed for the summarized data retrieval. In the first part, variables such as year of publication, study design, setting and data sources were retrieved. The second part retrieved cost-related data. Historical conversion rates were based on average conversion rate per year for that particular financial year (MYR to USD). The third part included items related to the evaluation of quality and methodological soundness of cost of illness (COI) studies. We utilized a checklist developed by Drummond et al.  for econThe initial search identified 385 records. From these, 52 records were duplicates and were removed. Of the remaining 333 records, 305 were excluded through screening of titles and abstracts, leaving 28 articles for full-text review. These 28 articles were screened manually and 3 additional articles known to authors were included. After assessing the full-text articles, 19 were excluded. A total of 12 studies were selected for final inclusion into this systematic review .n = 8) of the studies were conducted by local researchers in healthcare facilities in Malaysia [Malaysia ,31,32,33Malaysia ,35,36,37Malaysia ,39 were Malaysia , two stuMalaysia ,29, one Malaysia  and two Malaysia ,32. The Malaysia ,30,34 ann = 10) estimated the cost of diabetes using the prevalence-based approach [Most common epidemiological approaches used in health economic evaluations are either the prevalence-based approach or the incidence-based approach . The preapproach ,33,36,37approach ,35.A global study estimated the cost of diabetes by using country-by-country expenditures . Six stun = 7) reported cost estimates from the healthcare system (provider) perspective [Various perspectives are used when analyzing health economic studies. The common ones include patient’s perspective, such as out-of-pocket payments, employer’s perspective like loss of productivity; health system perspective, like evaluating costs of hospital and primary healthcare (clinic services); government’s perspective, such as estimating the infrastructure or health promotion/support programs; and lastly, the societal perspective to evaluate income loss during the care for the sick ,42. In tspective ,35,36,37spective ,28,30,33spective .The single global study that estimated country-by-country expenditures for diabetes utilized the sum-all medical approach . Cost anOf the six studies that reported the costing components to estimate direct costs, two studies estimated the costs for outpatient follow-up visits, two looked into inpatient costs, three reported drug costs, including drugs for diabetes complications and another three reported on estimated costs of managing diabetes complications.Four studies that evaluated capital and recurrent costs also reported the respective costing components. Capital costs that included buildings, equipment and vehicles were reported for all studies. Similarly, for recurrent costs, one study estimated operation and maintenance costs for building, one study reported operation and maintenance costs for vehicle, one study reported on training cost, three studies reported staff emoluments, two studies reported on investigation costs, three studies reported on drug costs, two studies reported on consumables costs and one study reported administrative, food and kitchen service costs.Of the twelve studies identified in this review, only two studies ,37 estimFour out of six studies that estimated direct costs were related to diabetes complications. One study reported that the management of end stage renal disease (ESRD)/nephropathy per patient per month on hemodialysis accounted for USD 795 and peritoneal dialysis accounted for USD 787 . AnotherOnly one study measured the indirect and intangible costs via a cost–benefit analysis approach for a diabetes self-management program . ThroughFour studies evaluated capital costs for diabetes management. All of these studies evaluated costs for buildings while three of the studies evaluated costs for equipment. Only one study estimated cost for a vehicle. For outpatient health clinic visits, average total capital costs per year ranged between USD 6.50  and USD Recurrent costs were evaluated in four studies ,27,28,32Of the twelve studies in this review, two studies described the factors associated with costs by using univariate analyses only ,36, two The results were clearly presented in most studies and consistently well reported in relation to the methods adopted . For sixCosts were discounted in four studies. In all studies, the discount rate was 3% ,29,34,35This systematic review is the first to compile all available reports on economic analyses of type 2 diabetes in Malaysia between 2000 and 2019. This study contributes to the understanding of type 2 diabetes cost burden in Malaysia and the methods used to evaluate these costs. The review records existing evidence in the literature on the overall costs of managing diabetes in Malaysia, the different costing components evaluated, the perspectives used and the factors that influenced cost estimates. A total of twelve studies were included: eight local studies, three Asian regional studies and one global study that reported country-specific estimates. Two studies attempted to generalize the cost estimates nationwide, while the remaining studies focused on cost estimation either from a single-center or multisite within certain states in the country.Type 2 diabetes has been a growing public health concern in Malaysia over the past two decades. The prevalence progressively increased from 8.3% in 1996 to 14.9% in 2006 and 17.5% in 2015 ,45, withThe Malaysian healthcare system is a two-tier system comprising public and private sectors, with voluntary private medical insurance coverage made available for the population. The public healthcare system is substantially subsidized by the government and the private healthcare system is accessible voluntarily by the population, mainly funded from out-of-pocket expenditure and insurance coverage. The medical card covers hospitalization and short-term follow ups after discharge and does not provide long-term outpatient care for insured persons. In 2017, the government subsidized 43.1% of the total national health expenditure (USD 13.6 billion), with 37.6% borne by the population via out-of-pocket expenditure. Nearly half (49%) or USD 4.8 billion of the out-of-pocket health expenditures were consumed by private hospitals, followed by private medical clinics (22%) and private pharmaceutical services (14%) . PrivateThe diabetes care model in Malaysia continues to evolve. Up to 80% of patients with diabetes sought care at health clinics 56%) or hospitals (24.6%) financed by the Ministry of Health, followed by private clinics (15.0%), private hospitals (2.5%) and self-medication (1.7%) [% or hospBurden from patients and societal perspective, such as loss of productivity due to diabetes-related complications and disability, needs to be studied extensively. Families with members who have diabetes bear a disproportionate share of burden in terms of reduced earnings and higher out-of-pocket expenditures . StudiesHalf of the economic burden studies in this review focused on diabetes-related renal diseases. There was only one national study that estimated the direct medical cost for selected macrovascular and microvascular complications. This study was based on the incidence rates from the UKPDS trial rather than incidence rates in Malaysia, which were lacking. Besides, additional costing studies on antidiabetic medications such as injectable therapies and newer oral agents, as well as medications for comorbidities for patients with diabetes, are needed to provide better estimates of the direct medical cost. Capital and recurrent cost estimates provide additional insights into planning of resource distribution. In the study by Ezat et al. , the totVariations in cost estimation models also pose substantial challenges in the quantification of economic burden. For instance, annual direct medical costs for hemodialysis estimated by Seng et al.  was USD Various factors have been found to be associated with direct costs of diabetes care, which include education, income, number of diabetes-related complications, duration of diabetes, treatment options, hospitalizations and surgery . In our The epidemiological approaches used in most studies were prevalence-based, which are mostly suited to measure costs related to chronic diseases such as diabetes. This finding was consistent with previous studies ,48,53. PThe current available literature on costs of diabetes and its related complications in this study provided a fragmented picture focusing on cost estimation from limited public healthcare facilities rather than the entire healthcare system in the country. The cost variation between rural and urban settings should also be addressed. Data biases and limitations in terms of contextualization of results also pose concerns in systematic reviews of economic burden of diabetes worldwide ,53,59. TThe national economic burden for type 2 diabetes was estimated to be USD 600 million, which was an underestimate based on limited studies. This underscores the need to develop comprehensive diabetes cost models to understand the economic burden of type 2 diabetes, which could form a platform to guide efficient resource distribution and planning of diabetes care in the population.


Orthobunyavirus, family Peribunyaviridae) is an arthropod-borne virus that causes congenital abnormalities in ruminants. Here, we report the complete genome sequences of two AKAV strains causing nonsuppurative encephalomyelitis in cattle by postnatal infection in Japan.Akabane virus (AKAV)  is an arthropod-borne virus that causes congenital abnormalities in ruminants. Here, we report the complete genome sequences of two AKAV strains causing nonsuppurative encephalomyelitis in cattle by postnatal infection in Japan.Akabane virus (AKAV) (genus  Akabane orthobunyavirus . Since it was first isolated from mosquitos in Gunma, Japan, in 1959, it has been widely detected in Australia, Asia, and Africa (Culicoides spp.) and causes spontaneous abortions, stillbirths, and congenital malformations in pregnant ruminants (Akabane virus (AKAV) is an enveloped, tripartite, negative-sense RNA virus of the species AKAV strains are clustered into four genogroups based on phylogenetic analysis of their medium (M) segments , 2. Genohttps://jorgensen.biology.utah.edu/wayned/ape), with the OBE-1 strain (the genogroup II strain that is the progenitor of the vaccine strain used in Japan) as the reference .Both virus strains were propagated in HmLu-1 cells. Their genomic RNA was extracted using the QIAamp virus RNA minikit . Reverse transcription-PCR was performed using SuperScript IV reverse transcriptase  and PrimeSTAR GXL DNA polymerase . Primer sequences are available on request. The 5ʹ- and 3ʹ-terminal sequences of all of the genomic RNA segments were determined using 5′ and 3′ rapid amplification of cDNA ends (RACE) . The seqThe full-length S, M, and L segments of KM-2/Br/06 are 856, 4,309, and 6,869 nucleotides (nt), respectively, whereas those of FI-1/Br/06 are 867, 4,308, and 6,867 nt, respectively. The GC contents of the segments were also determined . The nucThe sequences determined in this study will be useful in identifying the virulence factors implicated in bovine postnatal encephalomyelitis.LC552047.1 (KM-2/Br/06 S segment), LC552048.1 (KM-2/Br/06 M segment), LC552049.1 (KM-2/Br/06 L segment), LC552050.1 (FI-1/Br/08 S segment), LC552051.1 (FI-1/Br/08 M segment), and LC552052.1 (FI-1/Br/08 L segment).The sequences are available in GenBank under the accession numbers