The recent pandemic of the novel coronavirus disease 2019 (COVID‐19) has caused huge worldwide disruption due to the lack of available testing locations and equipment. The use of optical techniques for viral detection has flourished in the past 15 years, providing more reliable, inexpensive, and accurate detection methods. In the current minireview, optical phenomena including fluorescence, surface plasmons, surface‐enhanced Raman scattering (SERS), and colorimetry are discussed in the context of detecting virus pathogens. The sensitivity of a viral detection method can be dramatically improved by using materials that exhibit surface plasmons or SERS, but often this requires advanced instrumentation for detection. Although fluorescence and colorimetry lack high sensitivity, they show promise as point‐of‐care diagnostics because of their relatively less complicated instrumentation, ease of use, lower costs, and the fact that they do not require nucleic acid amplification. The advantages and disadvantages of each optical detection method are presented, and prospects for applying optical biosensors in COVID‐19 detection are discussed. Fast facts at the frontline: Optical biosensors have led to significant advancements in virus detection and imaging capabilities. Coupled with advanced instrumentation, they have enabled higher sensitivities while increasing the rate at which samples can be tested. These techniques can be developed into point‐of‐care (POC) diagnostics for viral detection and are promising alternatives to detect COVID‐19. The novel coronavirus disease 2019 (COVID‐19) has caused an unprecedented surge in virus research, in particular improving testing and diagnostics. The rapid global spread of COVID‐19, caused by the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) and slow and sometimes inaccurate testing has highlighted the need for more advanced imaging and detection techniques. The current standards for virus imaging include computed tomography (CT), single photon emission computed tomography (SPECT), and positron emission tomography (PET).Optical biosensors present an alternative method for virus detection due to their safe, straight‐forward use, and cost‐effective technology, including eliminating the need for nucleic acid amplification.ex vivo diagnostics of processed biological samples, direct imaging without sample preparation has many challenges: the presence of dense tissues in biological samples introduces loss of light directionality that results in a higher degree of scattering.Optical biosensing can combine detection and imaging which can provide a deeper understanding of a pathogen in addition to detecting it in biological samples.Fluorescence‐based sensing and imaging offers unique advantages such as good sensitivity, high temporal resolution, availability of biocompatible imaging agents, and noninvasive characteristics that make it relevant in research and clinical settings.Fluorescent biosensors have various parameters like intensity, energy transfer, lifetime and, quantum yield that can be exploited for virus detection.The fluorescence emission of sensors can be classified as up‐converting or down‐converting based on their excitation and emission wavelengths. Up‐conversion is when the emitted wavelength is shorter than the excitation wavelength (anti‐Stokes shift). Up‐conversion of the above mentioned NIR 1 excitation wavelengths to shorter visible wavelengths enables minimal autofluorescence, deeper sample penetration depth, higher signal‐to‐noise ratio, and high chemical and physical stability during biosensing.in vitro.in vivo. In another study, a bionic assay was developed for thrombin activity detection  based on peptide‐modulated CdTe QD aggregation, where the surface charge of CdTe QDs is regulated by the hydrolysis of a thrombin substrate peptide.in vivo is a major concern.There are multiple different light‐emitting materials (fluorophores) used in fluorescence‐based optical biosensors including inorganic semiconductor quantum dots (QDs), carbon dots (CDs), graphene nanostructures and organic conjugated polymer nanoparticles as shown in Figure H‐pyran (DCM) dye molecules  NPs  are macromolecules containing backbones with extended π‐conjugated structural units resulting in enhanced light‐harvesting and effective transport of excitons compared to organic dyes. They are an important class of materials due to their signal amplification in therapeutic activitiess Figure C.[37] s Figure D.[38]Surface plasmons are collective oscillations of the electron cloud at the surface of a metal excited by incident electromagnetic radiation. There are different types of surface plasmons that are associated with different metal structure types. For example, propagating surface plasmon polaritons (SPPs) are supported by metal films while localized surface plasmon resonances (SPRs or LSPRs) are supported by metal NPs. One of the drawbacks of planar surface plasmon sensors based on SPPs is their extended surface area that requires relatively large sample volumes and large numbers of molecular interactions to generate a detectable signal. A logical approach to overcome these limitations is the use of plasmonic NPs, which exhibit a reduced surface area compared to a metallic film.http://biosensingusa.com) and Carterra (https://carterra‐bio.com), which detects antibodies, single cells, viruses, proteins, and drug molecules. Ashiba et al. employed a dual antibody trapping system to detect norovirus by adding antibody functionalized QDs to a biosensor modified with virus antibodies.SPR detection can be modulated on the basis of changes in intensity,Plasmon‐enhanced fluorescence (PEF) was first investigated in the 1970s by Weber et al. and Knoll et al. using high‐quantum‐yield dye molecules.6 when the sample is located on or near nano‐textured surfaces of plasmonic metals.The discovery of the SERS effect in the 1970s demonstrated that Raman scattering efficiency can be enhanced by factors of up to 103O4 and CeO2) and carbon nanotubes can produce a color change due to catalyzing the reaction of a peroxidase substrate or their intrinsic peroxidase activity, respectively.[5 PFU or 0.2 HAU, similar to the rapid antigen test kits  unique to a specific virus as shown in Figure Microfluidics‐based LOC devices have been identified as lead candidates for in‐field POC diagnostics due to their ease of use and low‐cost analysisin vivo analysis of lung virus infiltration using a multicolor two‐photon imaging device for influenza virus in mice.x,y,z planar positions to determine when a single virus particle is interacting with a cell surface. They were able to successfully capture a single virus particle diffusing in the aqueous solution and landing on the cell plasma membrane. This work is extremely useful for studying the delivery of nanoparticles or therapeutics into cells. Studying viruses in their physiological environments, provides insight into host response mechanisms assisting in developing more direct therapeutic targeting.Nano‐biosensors are especially useful as site specific imaging tools to detect where virus particles reside, which is useful for assisting with therapeutic delivery tactics. Recently, Ueki et al. published the first protocol for 7 copies of the RdRp‐COVID sequence, which is higher than the viral load of SARS‐CoV‐2 virus soon after onset (1×106 copies/mL).The race to design a novel biosensor that would optimize COVID‐19 tests in terms of cost, rate of testing and sensitivity is underway. Although imaging‐based biosensing techniques are more expensive and complicated as a method for virus detection, they are useful in providing insight into better understanding the virus replication pathway and can help in developing treatment options. Biosensors have a unique capability for surface modification to create targeting sensors through attachment of biomolecules that detect specific viral sequences. SARS‐CoV‐2’s DNA‐RNA hybridization is used in RT‐PCR amplification and can be induced by thermal exposure at a temperature slightly lower than the nucleic acid strand melting temperature.www.capitalbiotech.com) uses similar diagnostics to screen for six common respiratory viruses; however, it requires 1.5 hours to generate results. Canon™  uses loop‐mediated isothermal amplification (LAMP), a similar nucleic acid amplification technique to PCR,[Several LOC diagnostic systems have received emergency use authorization (EUA) from the U.S. Food and Drug Administration (FDA).e to PCR,109 to gA lateral flow immunoassay (LFIA) was developed by Chen et al. to test for the presence of anti‐SARS‐CoV‐2 Immunoglobin G (IgG) antibodies in human serum.www.acrobiosystems.com) and the limited supply and high cost of SARS‐CoV‐2 spike antibodies , it has the potential to be used as an at home test for SARS‐CoV‐2. Similar strategies using fluorescence can be implemented using the previously described sandwich method by trapping the virus particle between a similar antibody conjugated NP and recombinant ACE‐2 protein attached to a surface. By designing the NPs using either gold or other SPR‐enhanced technology, the amount of sample necessary to provide a positive readout can be minimized. This reduces the amount of recombinant ACE‐2 protein and SARS‐CoV‐2 spike antibodies compared to the amount necessary to cause a colorimetric change, thereby minimizing cost. Ideally, this system can be designed with a washing step to remove the bound virus particles to prepare a reusable sensor.Of particular interest is the biophysical property of the SARS‐CoV‐2 virus to recognize and bind to angiotensin‐converting enzyme 2 (ACE‐2) at a high affinity.There has been extensive research on optical detection of virus particles by sandwiching them between either primary and secondary antibodies or between a peptide sequence and an antibody. Recent studies by Wu et al. and Chen et al. have provided evidence for antibodies that specifically bind to SARS‐CoV‐2.COVID‐19 has had a dramatic effect on the world, causing disruptive stay‐at‐home orders and an unprecedented surge in hospital demand for both treatment and diagnosis. Science communities have been tasked with developing new methods for detection and therapeutics for SARS‐CoV‐2. Facilities with RT‐PCR equipment have been inundated with samples for analysis; however, lack of sufficient RT‐PCR equipment and experienced personnel limit the number of samples that can be run. This has resulted in RT‐PCR being a major bottle‐neck for COVID‐19 testing. There is a growing need for novel biosensors that can match the sensitivity and selectivity of conventional nucleic acid tests. Nanomaterial‐based biosensors present ideal alternatives due their advancement in design and fabrication methods, high selectivity, low sensitivity, and good reproducibility. Any biosensor used for COVID‐19 and future viral outbreaks should be readily accessible, affordable, and ideally used either as an at‐home test or with minimal sample preparation. Optical biosensors provide additional and alternate methods of testing that have the potential to generate results at a much quicker rate than the tests that are currently being used for COVID‐19 testing. This minireview highlighted the current trends in optical biosensors and their ability for virus detection. Novel viral outbreaks will occur in the future, and it is necessary to have virus sensing technology in place to help aid in detection and to reduce virus spread.The authors declare no conflict of interest.Hemanth Maddali received his integrated masters degree from the University of Hyderabad in 2015. During his Masters, he worked on organic photonic micro‐resonators in Prof. Chandrasekar Rajadurai's lab. He then moved to NCBS, Bengaluru for an internship, where he worked with Dr. Sunil Laxman on mass spectrometry‐based analysis of metabolomics. He then moved to Rutgers to pursue his PhD in chemistry under the supervision of Prof. Deirdre O'Carroll with research focusing on doped conjugated polymer thin films for various optoelectronic devices.Catherine E. Miles received her bachelor's degree in chemistry from Western Washington University in 2016 where she worked in the lab of Prof. Mark Bussell focusing on developing catalysts for the removal of sulfur and nitrogen impurities from crude feed oil. She then moved to Rutgers University to pursue her PhD in chemistry under the guidance of Prof. Joachim Kohn, where she studies the effect of structure‐property relationships on drug delivery and degradation of tyrosine‐derived polymer microparticles.Joachim Kohn, PhD, FBSE leads the Laboratory for Biomaterials Research at Rutgers University. Kohn has made seminal contributions to the design, synthesis, characterization and fabrication of new biomaterials for regenerative medicine, tissue engineering and drug delivery. He pioneered the use of combinatorial and computational methods for the optimization of biomaterials for specific medical applications. Medical devices  are based on polymers developed by Kohn and have been implanted in about 500 000 patients in 46 countries.Deirdre M. O′Carroll is an Associate Professor in the Departments of Materials Science & Engineering and Chemistry & Chemical Biology at Rutgers University. Prior to joining Rutgers, she conducted postdoctoral research in plasmonics at California Institute of Technology in the US and at the University of Strasbourg and CNRS in France. She is a recipient of a National Science Foundation CAREER Award (2016) and an American Chemical Society Young Investigator Award (2017). Her research areas include nanophotonics and polymer semiconductors.


The common marmoset is widely used in current biomedical research for various research fields. We observed macrocytic anemia in a perinatal common marmoset with gradual weight loss and diarrhea. The objective of this case report is to describe the diagnosis and treatment of macrocytic anemia in a perinatal common marmoset.A 7-year-old female common marmoset showed clinical signs of gradual weight loss and intermittent diarrhea beginning 3 months after giving birth. Macrocytic anemia was diagnosed due to a decreased red blood cell (RBC) count, low hemoglobin level, and increased mean corpuscular volume (MCV). Multivitamins containing cobalamin and folate were administered for 7 days, and the patient’s RBC count recovered to near the normal range with this treatment.Macrocytic anemia can be diagnosed by evaluating the MCV on a complete blood count (CBC) and cobalamin or folate levels and be treated by supplementation with cobalamin and folate. Such supplements may be needed during pregnancy and lactation in female common marmosets and/or in animals with chronic diarrhea. Callithrix jacchus) are New World nonhuman primates (NHPs) that have been widely used for biomedical research in neuroscience, aging, reproductive biology, behavior, and drug development and safety because of they are small in size, cost relatively little to keep, require little effort for husbandry, are easy to handle for routine clinical procedures, have rapid reproductive turnover, and have a lower incidence of zoonoses than other NHPs such as macaques or baboons [Common marmosets  of red blood cells (RBCs) and is induced by cobalamin (vitamin B12) or folate (vitamin B9) deficiency. The objective of this case report is to report the diagnosis and treatment of macrocytic anemia in a perinatal common marmoset.®, Hi Well Healthcare, Auckland, New Zealand, Table The animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of the Biomedical Research Institute at the Seoul National University Hospital -accredited facility; IACUC number: 20-0161-S1A1). We obtained a group of common marmosets from CLEA . A 7-year-old female common marmoset ID: 17Cj03) showed clinical signs of gradual weight loss (from 306 to 250 g), intermittent diarrhea, depression, and a poor hair coat beginning 3 months after giving birth. A complete blood count (CBC) and biochemical analysis were performed for diagnosis  and non-megaloblastic. The megaloblastic form is due to impaired DNA synthesis from folate and/or vitamin B12 deficiencies, while the non-megaloblastic moiety (absence of hypersegmented neutrophils) occurs from multiple mechanisms such as alcohol consumption (RBC toxicity), hereditary spherocytosis (impaired volume regulation increases red cell size), hypothyroidism and liver disease (due to lipid deposition in the cell membrane), and marked reticulocytosis from states of excess RBC consumption such as hemolysis or turnover in pregnancy or primary bone marrow disease [The megaloblastic form of macrocytic anemia is induced by a low serum cobalamin or folate level. Evaluation of serum cobalamin and folate levels requires 0.8 ml of serum (1.6 ml of whole blood) in our test protocol. This is a relatively large blood volume to collect in a common marmoset, especially in an anemic animal. Thus, to evaluate serum cobalamin and folate levels, we alternatively selected an archived sample that was collected from a euthanatized 7-year-old female common marmoset (ID: 15Cj08) with clinical signs of gradual weight loss (from 269 to 224 g), chronic diarrhea, anemia, and the same history of disease onset after childbirth. This marmoset’s cobalamin and folate levels were 133 pg/ml and > 120 ng/ml, respectively . The reference ranges for these parameters from Parambeth et al.’s group were 322–2642 pg/ml for cobalamin and 54.8–786.4 ng/ml for folate [Another reason for low cobalamin and folate levels is chronic diarrhea. Intestinal absorption of cobalamin occurs via several processes, and absorption of a complex of cobalamin and intrinsic factors occurs through specific receptors in the enterocytes of the ileum . Ileal dIn conclusion, macrocytic anemia can be diagnosed by evaluating the MCV on a CBC and cobalamin or folate levels and be treated by supplementation with cobalamin and folate. Such supplements may be needed during pregnancy and lactation in female common marmosets and/or in animals with chronic diarrhea.


There has been an increased interest in conducting healthcare economic evaluations. Also, orthodontic treatments have gathered focus from an economic point of view, however orthodontic research seldom examines both clinical and economic outcomes.To evaluate and compare the costs of three retention methods: a bonded retainer to the maxillary four incisors, a bonded retainer to the maxillary four incisors and canines, and a removable vacuum-formed retainer (VFR) in the maxilla. The null hypothesis was that there was no difference in costs for the three types of retention methods.Three-arm, parallel group, single-centre, randomized controlled trial.Ninety adolescent patients, 54 girls and 36 boys, treated with fixed or removable retainers in the maxilla, were recruited to the study. The patients were randomized in blocks of 30, by an independent person, to one of three groups: bonded multistranded PentaOne (Masel Orthodontics) retainer 13-23, bonded multistranded PentaOne (Masel Orthodontics) retainer 12-22, and removable VFR. A cost analysis was made regarding chair time costs based on the costs per hour for the specialist in orthodontics, and material costs plus any eventual costs for repairs of the appliance. Changes in Little’s irregularity index and in single contact point discrepancies (CPDs) were measured on digitalized three-dimensional study casts. Data were evaluated on an intention-to-treat basis. The analysis was performed at 2 years of retention.No statistically significant difference in costs between the maxillary fixed retainers and the VFRs was found, however, the material and emergency costs were significantly higher for the VFR compared with the bonded retainers. All three retention methods showed equally effective retention capacity, and no statistically significant differences in irregularity or CPDs of the maxillary anterior teeth in the three groups was detected.It was a single-centre trial, and hence less generalizable. Costs depended on local factors, and consequently, cannot be directly transferred to other settings.All three retention methods can be recommended when considering costs and retention capacity.NCT04616755. Relapse is an unwanted side-effect of orthodontic treatment, both for the patient and the orthodontist . A partiThere are different retention strategies to maintain the position of the teeth in the maxilla. The strategies commonly include a bonded retainer between the maxillary canines , 7. AlsoRecently, there has been increased interest in conducting healthcare economic evaluations. The main factor is that resources  within the health sector are limited . While oWith resources within dental care being restricted, knowledge of effects and related costs are crucial when making treatment decisions. Thus, lack of economic analysis in dental health service may cause unsustainable over expenditure and result in decrease of services or resources in other areas of dental healthcare . RegardiRecently, we conducted a randomized controlled trial (RCT) on the stability of maxillary anterior teeth, comparing two bonded and a VFR after 2 years of retention . All thrAdolescent patients treated with fixed appliance in the maxilla and mandible, or solely in the maxilla, were recruited at the Orthodontic Clinic Växjö, Public Dental Service, Region Kronoberg, Sweden. Patients with clefts or syndromes, patients with agenesis or extracted maxillary anterior teeth, and patients who underwent orthognathic surgery, were excluded. Enrolment started in October 2013 and ended in October 2017. The last follow-up of the retentions was completed in October 2019. The trial was a single-centre, RCT with three parallel arms and a 1:1:1 allocation ratio. The Regional Ethical Research Board, Linköping, which follows the Declaration of Helsinki, approved the trial (Dnr 2013/131).After informed consent from each patient and their custodians was obtained, the participants were randomly allocated to one of the three retention groups as follows: 1. bonded retainer to the maxillary four incisors and two canines, 2. bonded retainer to the maxillary four incisors 12-22, and 3. removable VFR covering all maxillary teeth.The randomization process was prepared by an independent person and carried out by three staff members not involved in the trial. The randomization used blocks of 30. Every new participant randomly picked a sealed opaque envelope and revealed their group assignment by opening the envelope. Recruitment continued until the total number of participants met the estimated sample size. Data on all participants were evaluated on an intention-to-treat (ITT) basis. Consequently, all randomized patients remained in the allocated group and were followed during the 2 years of retention.Before the retention period, the patients were treated with a pre-adjusted fixed appliance in the maxilla, or in the maxilla and the mandible . The treatments were ended when the treatment goal was achieved, thus the overjet, overbite, and occlusion had to be normalized before the retention period was started. Records of the patients included study casts produced before orthodontic treatment (T0), after removal of the fixed appliance (T1), and after 2 years of retention (T2). The retention check-ups at the orthodontic clinic were performed at three scheduled occasions: 1 month, 1 year, and 2 years after insertion of the retainers. All three types of retainers were manufactured by dental technicians outside the clinic.The retainers in group A  covered all maxillary teeth . The VFRThe outcome measures were: 1. Total mean costs as chair time costs based on the costs per hour for the specialist in orthodontics; 2. material costs and any additional costs for repairs of the appliance. The post-treatment changes in irregularity of the six maxillary anterior teeth, according to Little’s irregularity index (LII)  and contWith results and consequences of each group’s retentions presumably being equivalent to each other, i.e. all three groups had retention during 2 years with the intention to stabilize the corrected maxillary anterior teeth, the difference between the groups was reduced to a comparison of costs.www.xe.com). The dental care including orthodontic treatment was funded by the publicly healthcare system, with a per capita compensation irrespective of type of retainer treatment.Orthodontic staff registered the chair time and whether the appointment was planned or an emergency visit. The treatment time costs and emergency costs including staff, room, room maintenance, dental equipment, and sanitation were based on the costs per hour for a specialist dentist (3400 SEK/€333) according to the price list of 2019 for specialist dentistry in the Public Dental Service in Region Kronoberg, Sweden. Based on the prices for the year 2019, the material costs were derived by the dental technician costs  plus any eventual costs for repairs of the appliance. All costs were expressed in Euros (€), SEK 100 = €9.9 at a mean currency value  and at the 2-year follow-up (T2). In addition, maximum CPD was assessed as the most severe single contact point per participant.3™ software  with semi-automatic segmentation  study casts for the three retention groups before treatment with fixed appliance (T0), start of retention (T1), and at the 2-year follow-up (T2). The study casts were digitized with a stationary 3D scanner  prior to the measurements. On the digital models, the measurement points were located using the OnyxCephentation .Data on all participants were evaluated on an ITT basis. Consequently, all randomized patients remained in the allocated group. Subsequently, patients with discontinued observation or lost to follow-up were still included in the final analysis by assessing the group’s maximum value for costs and cost changes, considering the primary outcomes  as well as for the secondary outcome variables representing the change in irregularity index and CPDs.α = 0.05, the sample size of each group was estimated to be 28. The sample size calculation for comparison of the retention capacity of the three retention methods has been published elsewhere  between the three retention groups. Consequently, using a power of 90% and lsewhere .post hoc test was used to compare the costs within and between the retention groups. Differences in LII and CPD between the three retention groups were tested as specifically described elsewhere (P < 0.05) were considered statistically significant.Data on costs were processed with the IBM-SPSS software . Analysis of variance with Tukey’s lsewhere . Continulsewhere . DiffereNinety patients, 54 females and 36 males, with a mean age of 15.9 years at start of the retention period, were recruited and the participants were followed for 2 years. All patients remained within the groups during the trial except one participant from group C (VFR) who changed domicile but, according to the ITT principle, was still included in the cost and retention capacity analysis The mean total retention cost per patient was €734 for the bonded retainer to the maxillary four incisors and two canines (13-23), €674 for the bonded retainer to the maxillary four incisors (12-22), and €778 for the VFR. No statistically significant difference in total cost was seen between the three retention methods .The mean cost for treatment time per patient was €633 for the bonded retainer to the maxillary four incisors and two canines (13-23), €600 for bonded retainer to the maxillary four incisors (12-22), and €579 for the VFR. No statistically significant difference between the retainers was found .P < 0.0001) , €49 for bonded retainer to the maxillary four incisors (12-22), and €83 for the VFR. The material cost was significantly higher for the VFR compared with the two bonded retainers ( 0.0001) .P = 0.005) (The mean emergency cost per patient was €50 for the bonded retainer to the maxillary four incisors and two canines (13-23), €25 for bonded retainer to the maxillary four incisors (12-22), and €116 for the VFR. The emergency cost was significantly higher for the VFR than for the two bonded retainers (= 0.005) .No statistically significant group difference was found in costs regarding absence from appointments. For the bonded retainer to the maxillary four incisors and two canines (13-23) as well as for the VFR, the cost per patient was €27 while the cost for the bonded retainer to the maxillary four incisors (12-22) was €11.All three retention methods showed good capacity to retain the maxillary anterior teeth. No statistically significant differences in irregularity of the maxillary anterior teeth in the three groups were detected. These data are specifically presented elsewhere .This trial was the first healthcare economic evaluation to assess costs of fixed and removable maxillary retainers and no difference in costs or retention capacities were found between the retention methods. Although, the material and emergency costs were significantly higher for the VFR compare to the bonded retainers, no statistically significant difference for the total retention costs was seen between the fixed retainers and the VFR, or between the two types of fixed retainers. Thus, the results of this trial confirm our hypothesis that there are no differences in costs between the three retention methods. Also, the three methods, without any difference between them, showed good clinical capacity to retain the maxillary anterior teeth; therefore, all three retention methods can be recommended both regarding retention capacity and costs.All the three types of retainers were manufactured by dental technicians outside the orthodontic clinic. It is conceivable that the material costs can be reduced if the retainers are manufactured at the clinic by a dental nurse or a dentist or even by a 3D printer. It was shown in the present trial that, even if the material and emergency costs were higher for the VFR, due to repeated damages or loss of the retainers, the total retention costs did not differ significantly between the three retainers. This might be explained by a shorter initial chair time at the clinic for the patients with VFRs compared with the patients with fixed retainers. Accordingly, the slightly higher treatment costs for the bonded retainers compared with the VFR equalizes the higher material cost for the VFR. The differences between the total costs of the different retention methods are not therefore significant.It can be pointed out that new techniques for removable retainers may impact costs and improve the ability to predict the outcome of the retention. Patient compliance can be improved by artificial intelligence or by devices such as thermo-sensitive microsensors. Their clinical application may reduce retention costs in the future , 25.per se implies that selection bias is avoided, and most importantly, any confounders were evenly distributed between the groups. In addition, the results were evaluated on an ITT basis and the attrition bias was low since there was only one drop-out in the trial.This study was implemented alongside an RCT regarding the stability of maxillary anterior teeth with fixed or removable retainers . The ranThe patients who were included in the trial were diverse in terms of gender distribution, age, and treatment at a specialist clinic, and thus, were considered representative of orthodontic patients. Hence, the design and performance of this trial allowed the results to be implemented in daily orthodontic practice.This trial was conducted at one orthodontic clinic which may involve a higher risk of bias compared with multi-centre studies due to a limited trial population, few operators, small number of involved staff members, and commendable organization .The number of patients who initially were not interested in being enrolled and participating in the trial was rather high, which therefore increased the risk of selection bias. The main reason for not enrolling in the trial was that these patients looked forward to having their treatment completed and not being further evaluated in a research trial.It is important to point out that monetary variables are influenced by local factors such as insurance systems, staff salaries, rental costs, taxes, urban versus rural areas, etc., and consequently, the figures shown in this trial cannot be directly extrapolated to other locations. Thus, the generalizability of the findings to other settings may be limited as the study was carried out on a regional scale.Another limitation is that the costs consisted of so-called direct costs. Hence, the trial did not include indirect costs, i.e. costs defined as loss of income  incurred by the patients’ parents’ absence from work to accompany the patient to the orthodontic appointment, including waiting time at the clinic and the travelling duration. However, the direct costs normally comprise the majority (65–90%) of the total costs , 28 and The use of maxillary fixed and removable VFRs showed no significant differences in costs, as well as good clinical capacity to retain the maxillary anterior teeth. Thus, all three retention methods can be recommended when considering costs and retention capacity.


The heterojunction has highly preferred orientation and exhibits well ferroelectric properties with perfect hysteresis loops and microscopic polarization switch behaviors. The most interesting thing is the abnormal dielectric relaxation phenomenon in the dielectric spectra at high frequency range and around the phase transition temperature of LSMO. By analyzing the resistance properties of LSMO films, it is indicated that charge-based interfacial coupling, Maxwell-Wagner effect due to the JT polaron and fast resistivity rise in LSMO layer is the main reason. This work emphasizes the crucial role of resistivity exchanges and of carrier accumulation at interfaces for the application of magnetoelectric heterojunction.The BaTiO BaTiO3 (BTO)/manganite heterojunction attracts much scientific and technological attention due to the excellent magnetoelectric coupling effect. The BTO/manganite can constitute a multiferroic tunnel junction which exhibits four or even eight resistance states in a single memory unit cell which can open a new pathway toward high-density nonvolatile memory [The multiferroic heterojunction composed of single-phase ferroelectric (FE) materials and ferromagnetic (FM) materials has great potential applications in next-generation information processing and storage devices, such as memory computer read heads, sensors, and multi-state memories ,2,3,4. Me memory ,2. The te memory ,6,7. On e memory ,9. Actuae memory ,11,12,131−xAxMnO3 , which is strongly correlated system with perovskite structure, has a strong interplay between electron transport, magnetism, and crystal lattice distortions and a rich carrier-density-temperature phase diagram. Among them, La0.7Sr0.3MnO3 (LSMO) not only exhibits ferromagnetic properties with high Curie temperature but also a metallic conduction at room temperature which can be served as the bottom electrode for the ferroelectric and dielectric properties measurements [In this work, BTO served as a ferroelectric constituent of the bilayer not only due to the archetype of ferroelectrics but also the sensitive physical properties ,15. The urements ,17. The 3 (STO) (100) single crystal substrate by sol-gel method. Lanthanum acetate (La(CH3COOH)3), strontium acetate (Sr(CH3COO)2), and manganese acetate (C6H9MnO6·2H2O) were dissolved into a solvent containing acetic acid (CH3COOH), deionized water and acetylacetonate to prepare the precursor solution. Barium carbonate (BaCO3) and titanium isopropoxide (C12H28O4Ti) were used as the sources for the precursor solution. The rotation speed and the spin time were at 5000 rpm and 30 s, respectively. After coating each layer, the film was fired at 200 °C for 180 s, then pyrolyzed at 400 °C for 180 s, and finally annealed at 850 °C for 600 s. Repeat the sinter process several times, and we finally get the BTO/LSMO composite film. The thicknesses of both LSMO and BTO layers are about 100 nm. The phase purity and crystal structure of the films were measured θ-2θ by high-resolution X-ray diffraction . Ferroelectric material test system  was used to characterize the ferroelectric properties. Piezoelectric atomic force microscopy  was used to characterize the microscopic ferroelectric domain properties. Dielectric properties were measured using a precision LCR meter  mounted on physical property measurement system  over the frequency range of 20 Hz–1 MHz within the temperature range of 10–400 K.The BTO/LSMO heterostructures were prepared on SrTiOθ-2θ patterns of BTO/LSMO bilayer composite films grown on STO (001) substrate. It is observed only the (00l) diffraction peaks of films and the substrates, which shows that the films have strong crystallographic texture and highly preferred orientation. The corresponding angles of BTO and LSMO diffraction peaks are 47.22° and 45.41°. The lattice constants in the out-of-plane direction are 0.3846 nm and 0.3992 nm for LCMO and BTO, respectively. Compared with the bulk LSMO (a = 0.3864 nm) and BTO (a = 0.3993nm), it is found that the out of plane strain of LSMO thin films is 0.4%, while the lattice constant of BTO film is almost constant, indicating that it is almost relaxation.r) and coercive voltage (Vc) of the thin film. When the external electric field is below the coercive field, the coercive field and remanent polarization increase significantly with the increase of the electric field. After that, the change becomes smaller with the applied voltage, which indicates the saturation of the polarization hysteresis loops. The maximum remanent polarization and coercive field were 8.5 μC/cm2 and 1.6 V, respectively.The microscopic piezoelectric and domain properties were analyzed by PFM. 6 Hz), and our films have upward self-poling and less domain wall, demonstrated by the PFM results. The LSMO have increasing resistivity with the increasing temperature, thermal carrier localized, and accumulated at the BTO/LSMO interface. This is consistent with the MW-type relaxation phenomenon. Therefore, we believe that the relaxation phenomenon observed in BTO/LSMO thin films may be due to MW effect with the carrier accumulation at the junction surface.The temperature dependences of dielectric properties of BTO/LSMO thin film at various frequency are shown in 3+-Mn4+ ions in octahedra and electrons recharging due to the balance of the strong interactions of double exchange, Jahn-Teller effect and Coulomb repulsion. The temperature dependence of the resistance of LSMO layer is shown in As the thicknesses of both LSMO and BTO layers are 100 nm, the local nanoscopic regions are formed with sufficient volume and thickness of LSMO, and the BTO/LSMO heterojunction consist of three layers: the BTO layer, the BTO/LSMO boundary layer, and the LSMO layer ,24. It mC1R2, with C1 and R2 corresponding to the larger capacitance and the smaller impedance of the two substances, respectively [C1 usually does not change with temperature, but R2 is closely related to temperature which is the main factor in the study of dielectric relaxation. Compared to the resistivity of BTO ~1012 W cm, the resistivity of LSMO is much smaller ~10−1 W cm, so the relaxation behavior of this heterojunction is closely related to the resistivity of LSMO layer. When the temperature rises, quick increasing of resistivity of LSMO is observed in the temperature and then causes the dielectric relaxation.Frequency dependent dielectric spectra of BTO/LSMO films at several temperatures is shown in ectively . In BTO/3/La0.70Sr0.30MnO3 (BTO/LSMO) bilayer magnetoelectric composite thin films were prepared by sol-gel method on STO substrates. XRD patterns show that the BTO/LSMO films have highly preferred oriented growth. The film shows good ferroelectric hysteresis loops, and the microscopic polarization reversal demonstrate the film has upward self-poling by PFM. The dielectric relaxation phenomenon (temperature around 270~300 K) was observed in the dielectric temperature and frequency spectrums and dielectric spectrum. The charge-related interfacial effect is the dominated factor and the intrinsic magnetoelectric coupling mechanisms can be excluded. The dielectric relaxation at high frequency may be related to not only MW effect but also Debye relaxation. Moreover, the electrical transport of LSMO film further proves that the dielectric relaxation behavior of the composite film is related to the resistance of LSMO due to the carrier accumulation at the junction surface. Our results of LSMO/BTO multiferroic composite films is very important for the applications of multiferroic materials in multi-state memory and other magnetoelectric fields.In conclusion, BaTiO


Scientific Reports 10.1038/s41598-021-81462-7, published online 21 January 2021Correction to: This Article contains a typographical error in the Introduction section where,Coronaviridae is a matter of global concern.”“SARC-CoV2, an enveloped single stranded positive sense RNA virus belonging to family Should read:Coronaviridae is a matter of global concern.”“SARS-CoV2, an enveloped single stranded positive sense RNA virus belonging to family


Togaviridae, Flaviviridae and Phenuiviridae were reported. Three viruses  were reported among the togaviruses whilst four  were among the flavivirus and only one virus, Rift Valley fever virus, was reported in the Phenuiviridae family. The majority of these mosquito-borne viruses were reported in Western and North-Western provinces. Aedes and Culex species were the main mosquito-borne viral vectors reported. Farming, fishing, movement of people and rain patterns were among factors associated with mosquito-borne viral infection in Zambia. Better diagnostic methods, such as the use of molecular tools, to detect the viruses in potential vectors, humans, and animals, including the recognition of arboviral risk zones and how the viruses circulate, are important for improved surveillance and design of effective prevention and control measures.Emerging and re-emerging mosquito-borne viral diseases are a threat to global health. This systematic review aimed to investigate the available evidence of mosquito-borne viral pathogens reported in Zambia. A search of literature was conducted in PubMed and Google Scholar for articles published from 1 January 1930 to 30 June 2020 using a combination of keywords. Eight mosquito-borne viruses belonging to three families,  Arthropod-borne viruses (arboviruses) are transmitted to susceptible vertebrate hosts by hematophagous, or blood-sucking arthropods such as mosquitoes, sandflies, lice, ticks and fleas . There aMedically important arboviral infections mostly include flaviviral infections such as yellow fever, Zika virus disease, West Nile fever, and dengue fever. In addition, Chikungunya fever caused by Chikungunya virus, an alphavirus has caused many documented outbreaks in Africa, Asia, Europe, the South Pacific and recently the Caribbean region ,4,5. TheAedes and Culex species play an important role in arboviral transmission. For instance, Aedes aegypti and Aedes albopictus have been incriminated in the transmission of prevalent arboviruses of medical importance. These include Chikungunya virus (CHIKV), dengue virus (DENV), and yellow Fever virus (YFV)  returned 95 articles. Altogether, the total number of retrieved articles was 560, of which 68 were systematic reviews, 33 were editorials and 94 were duplicates, and thus were removed. A total number of 365 articles were subjected to title and abstract screening against the inclusion/exclusion criteria. Of these, 332 articles were excluded because the articles were not conducted in Zambia (n = 33) were further subjected to full-text screening against the inclusion criteria by two investigators. After the full-text screening, sixteen (n = 16) articles were within the inclusion criteria and were thus included in the present systematic review. The searching process is summarized in The articles which passed the title and abstract screening  were cross-sectional studies ,25,26,27The studies obtained in our search used different study designs as well as sampling strategies to obtain data on the different mosquito-borne pathogens. The study designs employed were cross sectional (12/16), randomized cluster (2/16), retrospective (1/16) and case reports (1/16), with the sampling strategies used included convenience (10/16), two-stage cluster sampling (3/16), multi-stage (1/16), stratified (1/16) and a case report (1/16).To determine association between prevalence of mosquito-borne viruses and some potential risk factors, bivariate, multivariate and Pearson’s correlation tests were employed in the different studies. More details on study characteristics are provided in Concerning geographical locations, seven of Zambia’s ten provinces reported mosquito-borne viruses. These include Copperbelt, Central, Eastern, Lusaka, North-Western, Southern and Western provinces. However, Western and North-Western provinces had the most common sites for mosquito-borne virus studies as reported in eight and ten studies, respectively.Togaviridae, Flaviviridae and Phenuiviridae were recorded. Four viruses amid the flavivirus were reported DENV, WNV, YFV, and ZIKV  to ascertain the potential risk of YF infection. The findings revealed a prevalence of 0.3% for long-term infection and 0.2% for recent YF infection in the two provinces, respectively . The disTwo studies conducted on humans on ZIKV were identified during the review. The first study was conducted in 2015 to determine the prevalence and the risk factors for Zika virus infection in the Western and North-western provinces of Zambia. Out of a total of 3625, 6.1% of participants had Zika virus antibodies. Factors such as age, indoor residual spray, type of roof and visiting Angola were associated with Zika virus infection in bivariate analyses . In 2019CHIKV was first reported in 1961 in Luanshya, Copperbelt Province during an outbreak where similar cases of febrile conditions of sudden onset with headache, photophobia, pain in the joints and muscles, sore throat, occasional cough, and a maculo-erythematous rash appearing between the second and sixth days were observed. Three samples out of 7 tested positive for CHIKV IgM antibodies . Since tAlphavirus, member of the Togaviridae family together with CHIKV, has been limited to Central and South America .The search strategy involved a combination of keywords (“Northern Rhodesia” OR “Republic of Zambia”) AND  AND .The articles retained were populated in RefWorks (2020) database manager, and duplicates were automatically removed. Titles and abstracts of retained articles were subjected to the inclusion and exclusion criteria.All primary studies which focused on the occurrence and distribution of mosquito-borne viral diseases in Zambia from 1 January 1930 to 30 June December 2020 published in peer-reviewed journals, in either English or French languages, were included. Demographic health surveys indicating the burden of mosquito-borne viral diseases were also considered.Studies published in languages other than English or French, or lacking extractable data or not explicit in methodology, and systematic review papers were excluded. Abstracts without full manuscript texts were also excluded.The primary outcome was the distribution of mosquito-borne viral diseases. The secondary outcomes were the risk factors influencing their distribution.Potentially eligible articles were selected and screened using their title and abstract by two independent reviewers (RV and LL). The articles were divided into 2 subgroups “included” and “excluded” using the set inclusion and exclusion criteria. The final inclusion was done by analysing the full texts of the included articles. When necessary, any disagreements were resolved by arbitration of the third reviewer (BF). Data was extracted using an extraction tool from the Joana Briggs Institute Reviewers Manual (2018) for prevalence studies. Information regarding the authors, location, study design, characteristics of the sampled population, diagnostic tests used, and specific results was extracted and entered into an Excel sheet.The data extracted was used to create a map of the distribution and/or occurrence of mosquito-borne viruses across the country using ArcGIS version 10.3 .A narrative summary of included studies was done by pooling the raw data with an emphasis on reporting their characteristics along with data extracted relevant to the review outcomes. Analysis of quantitative studies was done based on the heterogeneity of the included studies.The findings of this review demonstrate that mosquito-borne viruses constitute a public health threat to the country. Despite this threat, very few studies have been conducted to understand the virus, vector and reservoir host interactions and dynamics. Though no epidemic has been reported as yet, favourable ecological factors noted in the country may lead to a rise in cases of mosquito-borne viral infections. Accurate information regarding the epidemiology and ecology of mosquito-borne viruses is of critical importance for implementing suitable surveillance strategies, prophylactic treatments, travel recommendations and clinical therapies. Furthermore, better detection methods, such as molecular tools, to detect the viruses in potential vectors, humans and animals, including the recognition of arboviral zones and how the viruses circulate, are important for improved surveillance and better appreciation of the impact of these viruses on animals and humans.


Hepatitis B virus (HBV) is a human pathogen that has infected an estimated two billion people worldwide. Despite the availability of highly efficacious vaccines, universal screening of the blood supply for virus, and potent direct acting anti-viral drugs, there are more than 250 million carriers of HBV who are at risk for the sequential development of hepatitis, fibrosis, cirrhosis and hepatocellular carcinoma (HCC). More than 800,000 deaths per year are attributed to chronic hepatitis B. Many different therapeutic approaches have been developed to block virus replication, and although effective, none are curative. These treatments have little or no impact upon the portions of integrated HBV DNA, which often encode the virus regulatory protein, HBx. Although given little attention, HBx is an important therapeutic target because it contributes importantly to (a) HBV replication, (b) in protecting infected cells from immune mediated destruction during chronic infection, and (c) in the development of HCC. Thus, the development of therapies targeting HBx, combined with other established therapies, will provide a functional cure that will target virus replication and further reduce or eliminate both the morbidity and mortality associated with chronic liver disease and HCC. Simultaneous targeting of all these characteristics underscores the importance of developing therapies against HBx. HBV is a major etiologic agent responsible for the development of chronic liver disease (CLD) which may resolve or progress to HCC. Although there are a variety of treatment options for patients with chronic hepatitis B associated HCC, prognosis and subsequent survival is poor, in part, because diagnosis is often made late, which limits the potential success of available treatments. Since the carrier state and progression of CLD are major risk factors for the development of HCC , alternaThe HBV encoded x antigen, HBx, is the virus contribution to the development of HCC. HBx contributes to pathogenesis in at least three important ways. First, HBx acts epigenetically to promote virus gene expression and replication . Second,An important reason for considering HBx as a therapeutic target has to do with the limitations of current therapeutic approaches. The goal of therapeutic intervention has been to reach a functional cure, defined as sustained off-treatment clearance of hepatitis B surface antigen (HBsAg) particles from blood, which occurs naturally in acute, resolving infections. However, this has not been achieved by any of the treatments currently available for patients with CLD. Initially, interferons (IFNs) and pegylated IFNs, which stimulate anti-viral immunity and block virus transcription and subsequent translation of virus proteins, were developed. Although virus resistance to IFN and pegylated IFN does not appear, these treatments were only effective in clearing HBsAg in only about 10–15% of HBV patients, require frequent intravenous administration, and often have serious side effects . IFN up-Other approaches aimed at preventing or curing infection include the development of inhibitory RNAs that block virus gene expression , and inhin vivo with low toxicity and that some act synergistically with nucleoside analogs in a preclinical animal model [in vivo, thereby trans-activating virus gene expression and replication [Additional compounds that inhibit or disrupt assembly of virus nucleocapsid particles or lead to the production of core particles that lack virus nucleic acids are also being pursued . The impal model . Capsid al model . Howeverlication , suggestlication , suggestlication , also suin vivo to promote virus replication [Part of the confusion concerning HBx, which has stunted interest in pursuing it as a therapeutic target, is that it seems to have a large number of reported activities, but it is not clear which activities are important to the biology of HBV . Anotherlication , the foclication . In thistrans-activates virus gene expression [Early studies supported the hypothesis that HBx was associated with virus nucleocapsids , that HBpression . HBx’s ipression , is critpression   that trigger and maintain auto-aggressive responses that persist even when virus replication becomes undetectable during stage 4. CLD progression may also reflect that HBx appears to protect infected hepatocytes from immune mediated destruction via up-regulation of nuclear factor kappa B  [Current drugs which target HBV replication have little or no impact upon the fragments of HBV DNA which become integrated into multiple sites within host chromatin during the regenerative process that takes place in the liver following a bout of hepatitis. These integrated HBV DNA sequences often encode the gene expressing HBx , and in tective) , and thatective) . HBx alstective) , 44 and tective) . Elevatetective) . HBx stitective) . In paratective) . Since Htective) , indicattective) , 49. If tective) . It is jtective) .The production of reactive oxygen and nitrogen species, perforins, granzymes and pro-inflammatory cytokines directed against virus infected cells from cell mediated immune responses trigger apoptosis. Cytotoxic T cells also trigger apoptosis via Fas signaling. Cytosolic HBx, stimulated by oxidative stress , over-riThe modulation of apoptosis by HBx appears to be context dependent. As outlined above, HBx could block apoptosis in CLD and via activation of NF-κB. In contrast, when HBx is expressed in normal mouse liver, and such mice are subjected to partial hepatectomy, liver regeneration is largely inhibited . In the trans-regulate the expression of cellular promoters, resulting in altered cellular phenotype. Some of the affected genes encode transcription factors that confer stem-like properties to infected hepatocytes, which include Nanog, Klf-4, Oct-3, and c-myc [It was discussed above that high intrahepatic concentrations of HBx originating from integrated viral sequences nd c-myc , 62. Impnd c-myc . In CLD,nd c-myc . Howevernd c-myc , which mnd c-myc , 65. Hignd c-myc , 65. If While mutations play a central role in carcinogenesis, epigenetic changes in host cell gene expression mediated by HBx may precede and promote the development of mutations. It is proposed that when HBx expression is high in infected liver, it down-regulates the expression of cyclin dependent kinase (CDK) inhibitors and the retinoblastoma gene product, Rb , as wellHBx also promotes the development of mutations by inhibiting DNA repair mechanisms. HBx binds to and inactivates poly (ADP-ribose) polymerase (PARP1), a crucial factor in DNA repair, by inhibiting recruitment of the DNA repair complex to the damaged DNA sites . HBx alsARF, p15INK4B, and p16INK4A, as well as the Rb gene had significantly higher methylation levels in HBV associated HCC compared to non-virus associated HCC [in vitro and its’ oncogenic properties in HBx transgenic mice. Thus, targeting HBx activity is expected to potentially decrease the accumulation of epigenetic changes and genetic instability/mutations in the infected cell [HBx epigenetically alters patterns of host gene expression through stimulation of histone deacetylases 1 and 2, as well as altered expression of DNA methyltransferases 3A and 3B . Genes eated HCC . These fted cell , therebyWAF1/CIP1, resulting in cell proliferation. HBx-upregulated miR-155 also represses HBV replication by modulating CCAAT/enhancer-binding protein (C/EBP) protein that activates the enhancer II/basal core promoter of the virus [HBx also impacts host gene expression by regulating the expression of multiple miRNAs. Differential miRNA expression alters the expression and activity of gene products that mediate various signaling pathways in the cell. For example, up-regulated miR-155 promotes β-catenin expression which, in turn, up-regulates c-myc . Up-reguhe virus .HBx-up-regulation of the miR-17-92 family stimulates cell proliferation by repressing the cyclin dependent kinase inhibitors p21, p27, and p57 . HBx-indin vivo [Efforts of the past few years have aimed at a “functional cure” for HBV, which is defined by the clearance of HBsAg from the blood. Given the persistence of integrated HBV DNA templates expressing virus proteins, and the difficulty in eliminating ccc HBV DNA, complete virus clearance has not been possible. This point is important because high levels of virus replication (with persistence of ccc HBV DNA) in patients with CLD contribute to the development of HCC . In the in vivo . Since Hin vivo , 85. By in vivo , 65, sugin vivo . Thus, iin vivo , 50.in vitro and in vivo [The discussion above strongly suggests that HBx is likely to be an important therapeutic target throughout the course of infection. However, there is no 3-dimensional structure for full length HBx, and it lacks functional motifs with characterized structural information from other proteins, so it has not been possible to use computer aided rational drug design . However in vivo . This ap in vivo , adminis in vivo . An HBx- in vivo . HBx spe in vivo , suggest in vivo . Given t in vivo . Alterna in vivo . The natAccumulating evidence suggests that HBx contributes to virus replication, protects infected hepatocytes against immune elimination, and contributes to multi-step hepatocarcinogenesis , 14 Fig. The facImportantly, chronic inflammatory diseases are often characterized by alterations in the composition of the gut microbiota, referred to as dysbiosis, which is accompanied by a “leaky gut” in which bacteria and bacterial metabolites contribute to hepatitis via portal vein transit . Studies


Enhanced production of reactive oxygen species (ROS) triggered by various stimuli, including viral infections, has attributed much attention in the past years. It has been shown that different viruses that cause acute or chronic diseases induce oxidative stress in infected cells and dysregulate antioxidant its antioxidant capacity. However, most studies focused on catalase and superoxide dismutases, whereas a family of peroxiredoxins (Prdx), the most effective peroxide scavengers, were given little or no attention. In the current review, we demonstrate that peroxiredoxins scavenge hydrogen and organic peroxides at their physiological concentrations at various cell compartments, unlike many other antioxidant enzymes, and discuss their recycling. We also provide data on the regulation of their expression by various transcription factors, as they can be compared with the imprint of viruses on transcriptional machinery. Next, we discuss the involvement of peroxiredoxins in transferring signals from ROS on specific proteins by promoting the oxidation of target cysteine groups, as well as briefly demonstrate evidence of nonenzymatic, chaperone, functions of Prdx. Finally, we give an account of the current state of research of peroxiredoxins for various viruses. These data clearly show that Prdx have not been given proper attention despite all the achievements in general redox biology. Viral infections are given more and more attention in recent years, especially during the pandemic of severe acute respiratory syndrome-associated coronavirus 2 (SARS-CoV-2) that was first reported in China at the end of 2019. ,4. SimilEnhanced production of reactive oxygen species (ROS) and dysbalanced antioxidant response that are often referred to as oxidative stress were shown to be the hallmarks of many viruses from various families. They include hepatitis B and C viruses, human immunodeficiency virus (HIV), various respiratory viruses ,7,8,9, h2O2 as a signaling molecule ,,78], 5 areatment . A ROS-ireatment . Howeverreatment ,75. Notereatment ,82. ThusExpression of Prdx6 is controlled by Sp1 and C/EBPβ transcription factors ,67 that Another transcription factor that controls the expression of several peroxiredoxins is FoxO3a that has multiple functions in a cell and whose functions are modulated by various protein kinases that affect its intracellular localization and activity . To dateThe roles of other transcription factors in controlling peroxiredoxin expression are less clear. It has been reported that Prdx3 and 5 can be induced during quercetin-triggered oxidative stress by the nuclear respiratory factor 1 (Nrf1) . NuclearThe role of peroxiredoxins in a cell is not restricted to the scavenging of peroxides, thus counteracting their enhanced production. They can exhibit regulatory functions by two mechanisms: Through specific oxidation of cysteine groups of other members of cell signaling pathways or by acting as molecular chaperones.2-Cys peroxiredoxins can form intermolecular disulfide bonds with a wide array of other cellular proteins, including transcription factors, protein kinases, and phosphatases, chaperones, metabolic enzymes/transporters, actors of the translational machinery, and many others . This is2O2 and passing oxidizing equivalents to the transcription factor, leading to the formation of disulfide bonds between monomers to form an oligomer with reduced transcriptional activity. This interaction is specific, as it occurs on membranes prior to oxidation of peroxiredoxin, and is further stabilized by the Annexin A2 protein . D. D128]. lication . Thus, Plication ,132,133.Prdx1 acts as an antiviral protein towards the hepatitis B virus—presumably by interaction with the HBx protein, as well as with viral RNA . It has Human papilloma and influenza viruses modulate peroxiredoxins to suppress oxidative stress in infected cells. In particular, the E7 protein of human papilloma virus 16 (HPV 16) triggers enhanced expression of Prdx2 . It coulPrdx1 is also a critical component of measles virus replication, as it interacts with the Mev-N protein, a component of RNA replicase . MoreoveFor respiratory syncytial virus (RSV), peroxiredoxins are required to protect the infected cells from virus-induced oxidative stress and concomitant cell death . As thisTo sum up, various viruses affect the expression of peroxiredoxins, although the data are nonsystematic and sometimes contradictory.Several viruses were shown to affect the expression of peroxiredoxins, although these data are rather nonsystematic. The uncontradictory data are presented in There is much discrepancy in data on the imprint of the influenza virus on peroxiredoxins, possibly due to different virus isolates and cell lines used by various groups. In the A/WSN/33 and (HPAI) H5N1 strains, the total amount of Prdx1 remains constant, whereas its levels at the membranes of A/WSN/33-infected cells increased , and in Flaviviridae family—the Zika virus was reported not to affect the expression of Prdx2, whereas the status of other peroxiredoxins, unfortunately, was not accesses [For flaviviruses, the situation is also not quite clear. Peripheral blood mononuclear cells (PBMC)s from patients with dengue virus exhibit enhanced expression of Prdx1 . Increasaccesses .Persistent lymphocytic choriomeningitis virus (PLCV) is a neglected pathogen that causes severe prenatal infection. It belongs to mammarenoviruses, as the abovementioned Junine mammarenovirus. PLCV was shown to downregulate the expression of several peroxiredoxins, including 2, 4, and 6 . A decreA different mechanism for the regulation of peroxiredoxin expression was observed for the human immunodeficiency virus, the protease of which recognizes Prdx2 as its substrate . HoweverThere are several reports on the association of changes in Prdx levels with some development of certain pathologies. In these cases, peroxiredoxins can be evaluated as biomarkers of viral infections or virus-mediated diseases. In particular, for patients with HIV—associated dementia (HAD), a decrease in Prdx3 levels in monocytes was shown, which could contribute to the development of neuronal damage through the loss of hydrogen peroxide scavenging capabilities . DecreasEnhanced expression of Prdx3 in livers of chronic hepatitis B patients with advanced fibrosis was also reported . An indePrdx1 can be used as a negative prognostic marker in patients with Dengue virus, i.e., of the development of hemorrhagic fever . Five-foPrdx 1 and Prdx2 are also released from the cells under lipopolysaccharide (LPS) treatment and influenza virus infection, adding them to a list of biomarkers of inflammation .Another piece of evidence was given by a very strong association of the rs7082598 single nucleotide polymorphism (SNP) in a gene encoding Prdx3 with cancer incidence in patients with human papilloma virus (HPV) . Thus, tIn the current review, we have briefly summarized the properties of all human peroxiredoxins, and thus, showed their significance in scavenging hydrogen and organic peroxides. In addition, a current understanding of the regulatory functions of these antioxidant enzymes has been demonstrated. These data demonstrate that peroxiredoxins are the proteins that can transfer signals from reactive oxygen species to specific proteins. However, the status of peroxiredoxins during various viral infections and their roles in the virus life cycle and development of virus-associated pathologies remains mostly obscure.To date, various viruses have been shown to trigger ROS production. However, almost all studies are based on a classical concept of oxidative stress—an imbalance between total ROS production and the antioxidant capacity of a cell. It hampers search for origins of ROS overproduction and does not identify cellular proteins that are controlled by specific cysteine oxidation—the subjects of modern redox biology. Since peroxiredoxins have the highest potential for peroxide neutralization, analysis of their oxidation in cells and their compartments in the future may reveal the exact sites for ROS production that are affected by infections. A single such study revealed oxidation of peroxiredoxins 1, 3, and 4 during RSV infection, suggesting the generation of ROS in their proximity . In addi


Pneumocystis jirovecii (PJP) and Moraxella osloensiswere detected. Moreover, in the presence of CMV, Pneumocystis jirovecii, Veillonella parvula, and other species showed dramatic changes in the lung of SOT patients, implying that high degree of co-infection between CMV and Pneumocystis jirovecii may occur. Taken together, our study shows that the presence of virus is associated with worse prognosis and dramatically altered lung microbiota in SOT recipients.Solid organ transplantation (SOT) is the final therapeutic option for recipients with end-stage organ failure, and its long-term success is limited by infections and chronic allograft dysfunction. Viral infection in SOT recipients is considered an important factor affecting prognosis. In this study, we retrospectively analyzed 43 cases of respiratory infections in SOT recipients using metagenomic next-generation sequencing (mNGS) for bronchoalveolar lavage fluid (BALF). At least one virus was detected in 26 (60.5%) recipients, while 17 (39.5%) were virus-negative. Among virus-positive recipients, cytomegalovirus (CMV) was detected in 14 (32.6%), Torque teno virus (TTV) was detected in 9 (20.9%), and other viruses were detected in 6 (14.0%). Prognostic analysis showed that the mortality of the virus-positive group was higher than that of the virus-negative group regardless whether it is the main cause of infection. Analysis of different types of viruses showed that the mortality of the CMV-positive group was significantly higher than that of the CMV-negative group, but no significant difference was observed in other type of virus groups. The diversity analysis of the lung microbiome showed that there was a significant difference between the virus-positive group and the negative group, in particular, the significant differences in microorganisms such as  Solid organ transplantation (SOT) is the final therapeutic option for recipients with end-stage organ failure, and its long-term success is limited by infections and chronic allograft dysfunction . For lunCytomegalovirus (CMV), the most common viral pathogen, historically has been associated with worse mortality in SOT recipients . Recent In this study, we aimed to comprehensively examine the real-world clinical impact of virus on the outcome in SOT recipients. Bronchoalveolar lavage fluid (BALF) samples were collected from SOT recipients with lung infections for mNGS. According to the results of mNGS, we analyzed the prognostic difference between the virus-positive group and the negative group and studied the contribution of different viruses to the prognosis. In addition, we analyzed the impact of virus on the microbiome of the lungs. Our results indicate that the virus is associated with a worse prognosis, whether or not it is the main cause of infection. Our results may have important implications for the clinical management and follow-up research in SOT recipients.The solid organ transplant recipients included in this study were all from Sichuan Provincial People’s Hospital. Our study passed the review of the Medical Ethics Committee of Sichuan Provincial People’s Hospital, and each enrolled recipient or their family signed a written informed consent. From November 2018 to September 2020, recipients with suspected infection were enrolled in this study following kidney, lung, or liver transplantation, and the recipients’ alveolar lavage fluid was collected from suspected lung infection recipients for mNGS analysis. The diagnosis of lung infection is based on the following: (1) There are related clinical symptoms, such as cough and sputum, difficulty breathing, fever, etc.; (2) There are related manifestations in imaging, such as pulmonary exudation; (3) Two or more SOT experts highly suspect lung infection with other content. Data collected on the enrolled recipients included body mass index (BMI), ICU length of stay, sequential organ failure assessment (SOFA) and Acute Physiology and Chronic Health Evaluation (APACHE II) scores, chronic disease history, and findings on other routine examinations.Samples were taken from recipients in two situations: BALF was collected when lung infection occurred during hospitalization, and β-lactam or carbapenem antibiotics were used for prophylactic treatment; when lung infection occurred after discharge, BALF was collected before anti-infective treatment. Samples of 1.5-3 mL BALF were collected according to standard procedures. A 1.5- mL microcentrifuge tube with 0.6-mL sample, enzyme, and 1 g of 0.5 mm glass beads was attached to a horizontal platform on a vortex mixer and agitated vigorously at 2800-3200 rpm for 30 min. Then the 0.3-mL sample was separated into new 1.5-mL microcentrifuge tubes and DNA was extracted using the TIANamp Micro DNA Kit  according to the manufacturer’s instructions. Then, DNA libraries were constructed through DNA fragmentation, end repair, adapter ligation, and PCR amplification. Agilent 2100 was used for quality control of the DNA libraries. Quality-qualified libraries were sequenced on the BGISEQ-50/MGISEQ-2000 platform. High-quality sequencing data were generated by removing low-quality reads, followed by computational subtraction of human-host sequences mapped to the human reference genome (hg19) using Burrows-Wheeler Alignment. After removal of low-complexity reads, the remaining data were classified by simultaneous alignment with four microbial genome databases, consisting of bacteria, fungi, viruses, and parasites.The survival of recipients within 90 days after transplantation was recorded, and the survival curve was made using GraphPad Prism 9. Comparison of survival curves was performed by the log-rank (Mantel-Cox) test.The analysis of alpha diversity was used to estimate complexity of taxonomic diversity for individual samples based on the Shannon index. The data matrix consisting of relative abundance for individual taxa across the samples was used for the Shannon index boxplot using R packages ggplot2 v3.3.2 and ggpubr v0.4.0. The visualization was implemented in the R environment v4.03.To assess the compositional similarity among the studied samples from different microbial communities, the Bray-Curtis measure of beta diversity was employed to compare all pairwise taxonomic relative abundances using an R function vegdist in the package vegan v2.5-7. Based on the resulting Bray-Curtis similarity distance matrix, non-metric multidimensional scaling (NMDS) was adopted to display the dispersion of community structure using an R function metaMDS in the package vegan v2.5-7. The scatterplot was implemented by using ggplot2 v3.3.2 in the R environment v4.03.To assess the compositional similarity among the studied samples from different microbial communities, the Bray-Curtis measure of beta diversity was employed to compare all pairwise taxonomic relative abundances using an R function vegdist in the package vegan v2.5-7. Based on the resulting Bray-Curtis similarity distance matrix, Principal Coordinate Analysis (PCoA) was adopted to display the dispersion of community structure using an R function cmdscale in the package vegan v2.5-7. The scatterplot was implemented by using ggplot2 v3.3.2 in the R environment v4.03.The statistical difference for the taxonomic profiles between study groups was evaluated using the function ANOSIM from the R package vegan v2.5-7. The similarity boxplot between groups was implemented using R package ggplot2 v3.3.2.Based on the taxonomic relative abundance profile, the heatmap for visualizing relative abundances of the top 20 taxa was created using pheatmap v1.0.12 in the R environment v4.03.Based on the taxonomic relative abundance profile, the boxplot of the top 20 taxa is created using ggplot2 v3.3.2 in the R environment v4.03.2. Of which, 32 recipients have been admitted to the ICU, average ICU stay was 8.8 days. There were 32 recipients evaluated by SOFA and APACHE II with average scores of 6.7 and 10.9, respectively. The most frequent chronic disease among the recipients was hypertension (17 cases), followed by hepatitis B viral infection (8 cases), diabetes, chronic obstructive pulmonary disease, and coronary heart disease  samples was performed in all SOT recipients to study virus and other microbial communities. At least one virus was detected in 26 (60.5%) recipients, while 17 (39.5%) were virus-negative. Among virus-positive recipients, cytomegalovirus (CMV) was detected in 14 (32.6%) recipients, and TTV was detected in 9 (20.9%) recipients, and other viruses were detected in 6 (14.0%) recipients, as shown in To observe the impact of virus on prognosis in these recipients, we evaluated the severity of disease (SOFA and APACHE II scores) and mortality (whether the recipient survived 90 days after transplantation). The results showed that there was no significant difference in SOFA and APACHE II scores between the virus-positive group and the virus-negative group. In this study, a total of 8 cases died within 90 days after transplantation, all of which were related to infection. However, in some cases, the virus may not be considered as the main cause of infection Table 2.Pneumocystis jirovecii, Moraxella osloensis, CMV) and other species beyond the top 20  and other species, as shown in Pneumocystis jirovecii.Considering that CMV is the main type of virus in our study (14/26) and its impact on mortality, we did a similar analysis of the microbiome between CMV-positive and CMV-negative groups. Diversity analysis and difference analysis showed that there was no significant difference between the CMV-positive group and the CMV-negative group, as shown in In this study, we explored virus detected in respiratory sample of 43 solid organ transplant recipients by mNGS. Viruses were detected in the bronchoalveolar lavage fluid (BALF) of 26 recipients. The most common type was CMV. The results showed that virus was associated with increased mortality in SOT recipients, and CMV may be the main virus. In addition, our results indicated that the virus may affect the composition of lung microbial species in solid organ transplant recipients.Unfortunately, although there are differences in mortality between the virus-positive group and the virus-negative group, the survival curve P value did not reach significance (p = 0.0828), which may be related to a small sample size. Previous studies have shown that CMV can increase mortality in SOT recipients . In thisDue to the immunosuppressive state, the infection of SOT recipients may be multi-pathogenic, and sometimes it is difficult to define a major pathogen. The 8 deaths in this study were all related to infection, of which at least one virus was detected in 6 cases, but in most cases the virus could not be considered as the main infectious agent. In order to check whether the death is related to other factors, we compared the age, BMI, and the proportions of chronic disease between the death group and the survival group, and the results showed no difference. Our results indicate that the virus is associated with a worse prognosis, whether or not it is the main source of infection. Previous related studies have judged the virus as the main pathogen and found that it leads to a worse prognosis. Our results indicate that the presence of the virus may be a marker of poor prognosis.In this study, why viruses that are not considered as the main pathogens are also related to high mortality may be a topic of our concern. In many cases, the presence of the virus, such as TTV, is considered to be related to the patient’s low immunity . One hypmNGS is the main detection technique in this study. At present, there are fewer methods to detect viruses, and there are fewer types of viruses that can be detected. mNGS can cover almost all viruses except RNA viruses, which can be detected by mNGS in the RNA process. Therefore, mNGS is a very effective method for multi-virus detection. The virus was detected in 26 of the 43 recipients in this study, with as many as 8 types of viruses. Each recipient had undergone CMV and EBV serological testing on admission, now the data of antibody testing is added in There have been some studies on the microbiome in SOT recipients, but most of them focused on the intestinal flora , and theMoraxella osloensis, one of the top 20 species in lung microbiology, decreased in the virus-positive group, and the proportion of Veillonella parvula, also one of the top 20 species in lung microbiology, decreased in the CMV-positive group. Moraxella osloensis is an aerobic gram-negative bacterium that is saprophytic on human skin and mucous membranes. It is also considered to be part of the normal flora of the human respiratory tract. The reported diseases caused by Moraxella osloensis infection include endocarditis, meningitis, osteomyelitis, septic arthritis, and bacteremia (Veillonella parvula is an anaerobic gram-negative bacterium and is considered to be a common bacterium in the oral cavity, gastrointestinal tract, and vagina. It has been reported as a pathogen related to meningitis, periodontitis, chronic maxillary sinusitis, sinusitis, osteomyelitis, bacteremia, pelvic abscess, and testicular epididymitis (In addition to PJP, our results also showed that virus affected the proportions of other microbes in the respiratory tract. For example, the proportion of cteremia . Veillonidymitis . PathogeOur study also has some limitations. Although the difference in survival curves between the virus-positive group and the negative group could be observed, it was not significant due to the small size of samples. For this same reason, only 6 cases were detected in other virus groups except CMV and TTV, which is meaningless in statistics. In this study, we only extracted DNA from BALF samples, so RNA viruses are outside the scope of our research. Three types of transplanted organs were enrolled in this study, but there were only 4 recipients of liver transplantation, no one died and one of them was virus-positive. So, the results of this study are more related to kidney and lung transplantation. Our research showed that virus had similar effects on mortality in kidney and lung transplantation. However, it is well known that the partial pulmonary microbiota of the recipient comes from the donor, which may undergo a complex reconstruction. Therefore, whether the impact of virus on the lung microbiome of lung transplant recipients is different from other SOT transplants needs more study and elucidation. Due to the BALF samples were collected by performing invasively endoscopy, the patients are unable to tolerate multiple collections during infections. Therefore, it is unlikely to monitor the change of microbiome at different time points in the patients. By realizing this difficulty, in this study, one time analysis of the microbiome in respiratory tract may not be able to demonstrate the dynamic changes.In summary, we studied the impact of virus on mortality and the lung microbiome in SOT recipients. The results showed that virus increased mortality regardless whether it is the main reason of infection, and CMV may be responsible for this. The results indicate that the presence of the virus may be a marker of poor prognosis. The proportion of PJP in virus-positive recipients increased significantly; the co-infection rate with PJP reached 71.4% in CMV-infected recipients. The results suggest that we should attach great importance to the co-infection with PJP when dealing with CMV infection. The mNGS results revealed the viral spectrum of SOT recipient lung infection, which can provide clinical reference. At present, there is a lack of detection methods for viruses, and mNGS has been gradually applied in clinical pathogen diagnosis. Our study explored the advantages of mNGS for viral infection and simultaneous detection of other pathogens. In addition, virus can also change the respiratory microbiome, and the differences in some background bacteria may provide references for subsequent research and clinical management.The raw data of mNGS in this study has been uploaded to the SRA database of NCBI, and the data is public. The project number is PRJNA806679.The studies involving human participants were reviewed and approved by the Medical Ethics Committee of Sichuan Provincial People’s Hospital. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the parents. All the authors listed have approved the manuscript that is enclosed.All authors contributed to the study conception and design. Research design and approval were completed by YK, XH, and YF. Recipient enrollment and management were completed by LP. Data analysis and the manuscript writing were completed by FW. Review and editing of manuscript were completed by LP, QC, ZX, and YF. Microbiome analysis were completed by ZX. Transplant and data collection were completed by HH, TT, RY, YH, and XZ. All authors contributed to the article and approved the submitted version.Authors FW, QC, ZX, and YF are employed by Genoxor Medical Science and Technology Inc.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Culex genus, but occasionally also through transfusion/transplantation of substances of human origin (SoHO) , percutaneous exposure or inhalation in laboratories, or transplacental passage from mother to fetus” was changed to “The virus is primarily transmitted through the bites of infected mosquitoes, mainly of the Culex genus, but occasionally also through transfusion/transplantation of substances of human origin (SoHO) , percutaneous or conjunctival exposure in laboratories, or transplacental passage from mother to fetus” in the article ‘Epidemiology of human West Nile virus infections in the European Union and European Union enlargement countries, 2010 to 2018’ by Young et al., published on 13 May 2021. This change was made on 20 May 2021. On request of the authors, the sentence “The virus is primarily transmitted through the bites of infected mosquitoes, mainly of the


Prokaryotic viruses, which infect bacteria and archaea, are the most abundant and diverse biological entities in the biosphere. To understand their regulatory roles in various ecosystems and to harness the potential of bacteriophages for use in therapy, more knowledge of viral-host relationships is required. High-throughput sequencing and its application to the microbiome have offered new opportunities for computational approaches for predicting which hosts particular viruses can infect. However, there are two main challenges for computational host prediction. First, the empirically known virus-host relationships are very limited. Second, although sequence similarity between viruses and their prokaryote hosts have been used as a major feature for host prediction, the alignment is either missing or ambiguous in many cases. Thus, there is still a need to improve the accuracy of host prediction.In this work, we present a semi-supervised learning model, named HostG, to conduct host prediction for novel viruses. We construct a knowledge graph by utilizing both virus-virus protein similarity and virus-host DNA sequence similarity. Then graph convolutional network (GCN) is adopted to exploit viruses with or without known hosts in training to enhance the learning ability. During the GCN training, we minimize the expected calibrated error (ECE) to ensure the confidence of the predictions. We tested HostG on both simulated and real sequencing data and compared its performance with other state-of-the-art methods specifically designed for virus host classification .HostG outperforms other popular methods, demonstrating the efficacy of using a GCN-based semi-supervised learning approach. A particular advantage of HostG is its ability to predict hosts from new taxa.The online version contains supplementary material available at (10.1186/s12915-021-01180-4). Prokaryotic viruses (shortened as viruses hereafter) play an important role in the microbial system dynamics. They regulate the ecosystem by limiting the abundance of their hosts through ongoing lytic infections. Because of the threat of antibiotic resistant pathogens, there is resurging interest of using phages as an alternative strategy to treat bacterial infections . A fundaHowever, despite its importance, the identified virus-host relationship is only the tip of the iceberg. The gap between the sequenced prokaryotic viruses and the known virus-host relationship is expanding quickly. Experimental methods, such as single-cell viral tagging , can detThere are three main challenges for computational prediction of the virus-host relationships. First, the known virus-host interactions are limited. One of the most widely used datasets, the VHM dataset , containSeveral attempts have been made to predict hosts for viruses based on the genomic sequences . They cak-mer-based features for prediction. VHM employs similar oligonucleotide frequency patterns between viruses and hosts and predicts the candidate host with the smallest distance for each input virus. PHP applies a Gaussian model to learn Gaussian distributions for the known virus-host pairs and outputs the probability for each input pair. Then, it uses the pair with the highest probability to assign a label for each virus. Unlike VHM and PHP, WIsH Fig S2 – [Associations between accuarcy and similarity at genus level. X-axis: Mash distance. Left Y-axis: Accuracy. Right Y-axis: number of testing virus.]Fig S3 – Tables S1-S2.Table S1 –[Parameters used for training CNN and GCN.]® Xeon® Gold 6258R CPU with 8 cores.]Table S2 – [Elapsed time for each tool. All the methods were run on Intel


Flaviviruses circulate worldwide and cause a number of medically relevant human diseases, such as dengue, Zika, yellow fever, and tick-borne encephalitis (TBE). Serology plays an important role in the diagnosis of flavivirus infections, but can be impeded by antigenic cross-reactivities among flaviviruses. Therefore, serological diagnosis of a recent infection can be insufficiently specific, especially in areas where flaviviruses co-circulate and/or vaccination coverage against certain flaviviruses is high. In this study, we developed a new IgM assay format, which is well suited for the specific diagnosis of TBE, Zika and dengue virus infections. In the case of TBE and Zika, the IgM response proved to be highly specific for the infecting virus. In contrast, primary dengue virus infections induced substantial amounts of cross-reactive IgM antibodies, which is most likely explained by structural peculiarities of dengue virus particles. Despite the presence of cross-reactive IgM, the standardized nature and the quantitative read-out of the assay even allowed the serotype-specific diagnosis of recent dengue virus infections in most instances. Flavivirus infections are of global importance for human health and pose increasing threats, as illustrated by the spread of dengue (DEN), Zika, West Nile, and tick-borne encephalitis virus (TBE) viruses . ThesThe diagnosis of flavivirus infections can be difficult, because the presence of the virus in the blood of patients (and correspondingly PCR positivity) is only transient in many instances . SpecWe assessed IgM specificity in flavivirus serodiagnosis with a newly developed IgM-capturing-ELISA format using a pre-formed detector complex with purified recombinant envelope (E) proteins of different flaviviruses. In our analyses, we performed a direct comparison of the cross-reactivity patterns in the following cohorts of recent flavivirus infections: 1. TBE patients in Austria, 2. Zika patients (travelers returning from endemic regions to Austria), and 3. dengue patients (travelers returning from endemic regions to Austria). Most of the Zika and dengue patients had a pre-existing immunity against TBE virus due to vaccination. As a control antigen for measuring broad flavivirus cross-reactivity, we used E from Rio Bravo virus . Dengue and Zika RNA TaqMan-based real-time PCR assays were carried out with primers and probes located in the 3’NCR and NS5, respectively, as described previously ,8. DenguSerum samples were analyzed with TBE IgM and IgG ELISAs based on purified TBE virus strain Neudörfl as described previously . BrieflyThe DENV DetectTM IgM Capture ELISA  and/or the SD Bioline Dengue Duo  were used for the detection of DENV-specific IgM antibodies according to the manufacturers’ instructions.Drosophila Expression System (Invitrogen) using the expression vector pT389 , encoding the E protein lacking the stem-anchor region , an enterokinase cleavage site and a double strep tag . As described previously [The recombinant soluble E proteins (sE) were produced with the eviously ,12, DrosMaxiSorp microtiter plates (Nunc) were coated overnight at 4 °C with rabbit immunoglobulin directed against human µ chains (Dako) ,11. SeruFor the detection of flavivirus-specific IgM antibodies, a preformed complex of strep-tagged sE and Streptactin-labeled horseradish peroxidase (HRP) was applied. sE proteins and Streptactin-HRP  were mixed in pre-determined optimal concentrations, incubated for 30 min at room temperature and either stored at −80 °C or directly used. The complex  was then added to the plates and incubated for 30 min at 37 °C. Absorbance was measured at 450 nm. Each serum was tested at least twice and mean absorbance values were calculated.The assay was validated with 38 flavivirus-negative diagnostic serum samples from previous studies . The cutp values ≤ 0.05 were considered significant.IgG ELISAs were carried out as previously described . BrieflyTBE, yellow fever (YF) and Zika NTs were performed as described previously ,15. BrieTo assess the patterns of IgM cross-reactivity after different flavivirus infections, we analyzed serum samples in the novel capture IgM E-complex ELISA from three groups of patients, in which a recent flavivirus infection had been diagnosed by routine serological assays and/or direct virus detection:Sixteen TBE patients . All werTwenty Zika patients . The serSixteen dengue patients . The sern = 5) and dengue (n = 2) patients had also been vaccinated against YF, as revealed by a positive YF NT; the single TBE-naïve dengue patient had a previous YF vaccination. The time points of vaccinations were not available to the diagnostic laboratory.Consistent with the high TBE vaccination coverage in Austria, 19 of the 20 Zika patients and 15 of the 16 dengue patients had been vaccinated against TBE in the past, as confirmed by TBE NTs . In addiTo investigate the specificity of IgM antibodies detected in the newly developed E-complex ELISA, we analyzed the 16 TBE, 20 Zika and 16 dengue samples as displayed in As displayed in For visualizing the capacity of the assay to discriminate between infections with different dengue virus serotypes, the IgM reactivity pattern of each dengue serum with the different antigens is displayed in Serum samples from two patients sent to our institution for dengue/Zika diagnosis yielded a pattern suggestive of a double infection with dengue and Zika viruses. Patient DZ1 a had retAs shown in Since most of the Zika and dengue patients had previous flavivirus vaccinations , we alsoIn this study, we show that a newly developed E-complex capture IgM assay allows a highly specific serodiagnosis of flavivirus infections. The response was type-specific for the infecting virus in the case of TBE and Zika virus infections, without detectable broadly flavivirus cross-reactive IgM antibodies. In contrast, substantial amounts of such IgM antibodies were found in dengue virus infections, as determined by their reactivity with the distantly related Rio Bravo virus E protein . These rDespite the induction of substantial amounts of cross-reactive IgM antibodies in dengue virus infections, differentiation from Zika virus infections (co-circulating in the regions from which the travelers returned) was possible in all cases analyzed . As expeNotably, the patterns of IgM cross-reactivity after dengue virus infections are quite divergent and apparently subject to strong individual variation . Some paDengue serotyping by IgM assays was also reported in previous studies, and it was found to be more reliable in the case of primary than secondary dengue virus infections ,25,26,27The importance of serological assays for the correct diagnosis of flavivirus infections, in particular IgM tests, is underlined by the fact that molecular techniques of virus detection in the blood are limited to the acute viremic phase, which is often missed at the time of first analysis . In the