Culex and Mansonia mosquitoes in the Zambezia province of Mozambique. Among the 1.7 and 2.6 million sequences produced from the Culex and Mansonia samples, respectively, 3269 and 983 reads were classified as viral sequences. Viruses belonging to the Flaviviridae, Rhabdoviridae and Iflaviridae families were detected, and different unclassified single- and double-stranded RNA viruses were also identified. A near complete genome of a flavivirus, tentatively named Cuacua virus, was obtained from the Mansonia mosquitoes. Phylogenetic analysis of this flavivirus, using the NS5 amino acid sequence, showed that it grouped with ‘insect-specific’ viruses and was most closely related to Nakiwogo virus previously identified in Uganda. Both mosquito genera had viral sequences related to Rhabdoviruses, and these were most closely related to Culex tritaeniorhynchus rhabdovirus (CTRV). The results from this study suggest that several viruses specific for insects belonging to, for example, the Flaviviridae and Rhabdoviridae families, as well as a number of unclassified RNA viruses, are present in mosquitoes in Mozambique.Mosquitoes carry a wide variety of viruses that can cause vector-borne infectious diseases and affect both human and veterinary public health. Although Mozambique can be considered a hot spot for emerging infectious diseases due to factors such as a rich vector population and a close vector/human/wildlife interface, the viral flora in mosquitoes have not previously been investigated. In this study, viral metagenomics was employed to analyze the viral communities in  Flavivirdae, Bunyaviridae, Rhabdoviridae, Togavridae and Reoviridae [Mosquitoes are important arboviral vectors with the potential to carry and spread pathogenic viruses to both humans and animals. It has been estimated that half of the global population is at risk of mosquito-borne viral infections and that these types of infections cause millions of deaths each year. Yellow fever virus, dengue virus, chikungunya virus, Japanese encephalitis virus and Rift Valley fever virus are examples of mosquito-borne viruses responsible for disease outbreaks in human and animal populations . Apart foviridae –7.The virus discovery rate in different species, such as mosquitoes, have increased through the availability of deep-sequencing technologies combined with viral metagenomic approaches. Apart from novel virus discovery, viral metagenomics have become a powerful method for genetically characterizing the complete viral populations of different sample , 3, 8, 9A total of 540 mosquitoes were collected in Cuacua (co-ordinates- S17°48.043' E 035°24.730), Zambezia province in Mozambique from October—November 2014 using CDC light traps. Individual mosquitoes were identified morphologically to determine the genus according to the descriptions of Edwards FW, 1941 and Hopkins GHE, 1952 , 12 and Each mosquito pool (up to 20 mosquitoes/pool) was mechanically homogenized using the Tissuelyser II (Qiagen) for 30 cycles/sec with 1 ml of TRIzol LS reagent (Invitrogen) and two 5 mm stainless steel beads. A centrifugation step (13000 g for 10 min) was performed, and the supernatant was collected for nucleic acid extraction. Total RNA was extracted from the TRIzol/mosquito homogenate according to the manufacturer’s instructions (Life Technologies), and the RNA pellet was dissolved in 40 μl of nuclease-free water. Five microliters of RNA from each pool was pooled, and the RNA was then concentrated and subjected to DNase treatment using RNeasy MinElute Cleanup kit (Qiagen) and RNAse-free DNase set (Qiagen). Ribosomal RNA was removed using a RiboZero kit (Ribo-Zero Gold (Human/Mouse/Rat), Illumina) according to the manufacturer’s protocol, and the RNA was again concentrated using RNeasy MinElute Cleanup kit (Qiagen).2, 2.5 mM deoxynucleoside triphosphates (dNTP), 0.8 mM FR20RV (5’GCCGGAGCTCTGCAGATATC’3) and 1.25 U AmpliTaq Gold DNA polymerase (Applied Biosystems). The PCR was performed with 10 min at 95°C; followed by 40 cycles of 30 sec at 95°C, 30 sec at 58°C, and 90 sec at 72°C; and a final elongation step at 72°C for 10 min. The tag-sequence was cleaved off with EcoRV (NEB), and the product was purified using a GeneJet PCR purification kit (Thermo Fisher Scientific).Ten microliters of RNA were used in the cDNA synthesis step, which also labelled the cDNA at the both ends with a tag-sequence. The first-strand synthesis was performed using the primer FR26RV-N (5’GCCGGAGCTCTGCAGATATCNNNNNN’3)  and SupeTM Chip v2 and 400 bp read length chemistry.The library preparation and sequencing of the amplified PCR product was performed at the National Genomics Centre, SciLifeLab, Uppsala, Sweden. An ion-torrent PGM sequencing platform was used with a Ion 318Anopheles, Aedes and Culex genomes with Bowtie2 [de novo assemblers using CodonCode Aligner 6.0.2 (CodonCode Corporation) and SeqMan NGen 11.2.1 (DNASTAR) to generate longer sequences. Alignment of contigs and unassembled reads to viral reference genomes was performed using CodonCode Aligner 6.0.2 (CodonCode Corporation).Raw data were filtered to remove sequences with low quality scores (Q<20) and exact duplicate reads. The ends of the reads with low quality were trimmed using PRINSEQ . The hos Bowtie2 . UnmappeViral contigs and single reads were used to manually design specific primers to confirm the presence of the virus as well as to amplify longer genome regions of the selected viruses from the original material. Thermal cycling was initiated with a denaturation step at 95°C for 10 min; followed by 35 cycles of 95°C for 30 sec, 58–60°C 30 sec and 72°C for 1 min; and a final extension at 72°C for 7 min. All the primers used are presented in To determine the phylogenetic relationship of the selected viruses from the mosquitoes, reference viral amino acid sequences representing flaviviruses and rhabdoviruses were obtained from GenBank. Sequence alignments were performed using ClustalW, and phylogenies were generated using the Neighbor-Joining method with MEGA 6.06 software , 17. TheCulex (156) and Mansonia (384) genera. A total of 1,733,788 and 2,623,031 sequences were obtained from the Culex and Mansonia pools, respectively  reference genomes. The majority of the remaining reads were classified as eukaryotic genome, mainly the host genome and as bacteria . Only 0.19% (3269 reads) and 0.03% of the reads (983 reads) were classified as viral sequences in the Culex and Mansonia mosquito pools. Viral sequences from Flaviviridae, Rhabdoviridae, Iflaviridae were identified; in addition, unclassified single-stranded and double-stranded viral RNA sequences were also detected  (GenBank KC505248.1) and Nakiwogo virus (NAKV) (GenBank GQ165809). In Mansonia spp., 175 reads showed highest similarity, at an amino acid level, to NAKV, which is a ssRNA positive-strand virus previously detected in Mansonia spp. in Uganda [Mansonia spp. mosquitoes, these sequences showed closest relationship to PCV  with an amino acid identity of 46–87%; the contigs were 310–880 nt in length), to Nienokoue virus  as well as to Culex flaviviruses and other mosquito flaviviruses. No sequences related to known pathogenic flaviviruses were identified in the data sets.In the current study, both the investigated n Uganda . The reaCulex and Mansonia spp. contained sequences related to rhabdoviruses, with 1% (36 reads) and 9% (89 reads) of all the viral reads in the respective data sets. In Culex spp., viral sequences were detected most closely related to Culex tritaeniorhynchus rhabdovirus (CTRV) (GenBank NC025384), with a sequence identity of 44–56% to the L segment (the RNA-dependent RNA polymerase gene). CTRV is an unclassified rhabdovirus identified in Culex tritaeniorhynchus mosquitoes from Japan in 2011 [Mansonia spp. showed closest relationship to CTRV. As for the Culex rhabdoviral sequences, the reads showed low sequence identity (40–50%) at the amino acid level with the L and G segments of CTRV. The reads were assembled to obtain longer viral sequences, and this generated 6 contigs that covered 95% of the CTRV G segment (Glycoprotein gene) and 60% of the L segment, represented as black bars in Mansonia spp. clustered together with CTRV in an unassigned group  to the polymerase protein gene.Both the with 1% 3 reads aned group . The rhaIflaviridae family were identified in Culex mosquitoes. These sequences were most closely related (24–63% amino acid similarity) to Nasonia vitripennis virus (GenBank FJ790487), Brevicoryne brassica picorna-like virus , Lygus lineolaris virus 1 (GenBank AEL30247.1) and Sacbrood viruses . No similarities were observed for Iflaviruses at nucleotide level.A large number of sequences (2870) related to the Culex spp., reads related to Dicistroviridae, Picornaviridae, Parvoviridae and unclassified ssRNA viruses were detected. A large number of sequences related to unclassified negative-strand ssRNA viruses and dsRNA viruses were identified in Mansonia spp.In addition to flaviviruses, rhabdoviruses and iflaviruses, other viral families were also detected in the two mosquito species . In CuleCulex and Mansonia), a large number of viral sequences were identified, some of which had close or distant relationships to viral families such as Flaviviridae, Rhabdoviridae and Iflaviridae etc. while others appeared to be associated with unclassified viruses.In the present study, viral metagenomics was used to identify mosquito-associated viruses in the Zambezia province of Mozambique. This is the first study where high-throughput sequencing has been used to explore the viral communities of mosquito populations in Mozambique. In the two mosquito genera . The detected novel rhabdovirus sequence also share similarity with Beaumont virus (43% at amino acid level) which was previously isolated in Australian mosquitoes [Rhabdoviruses are present in a wide range of hosts, including different plants, vertebrates and insects. Currently, there are 17 recognized and proposed genera present in the family, and many rhabdoviruses await taxonomic classification . Becausesquitoes . Phylogesquitoes , howeverThe current findings are in agreement with the previous studies and demonstrate that mosquitoes harbor a rich and diverse virome , 3, 41. S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Cytocompatible polysaccharide-based functional scaffolds are potential extracellular matrix candidates for soft and hard tissue engineering. This paper describes a facile approach to design cytocompatible, non-toxic, and multifunctional chitosan-cellulose based hydrogel beads utilising polysaccharide dissolution in sodium hydroxide-urea-water solvent system and coagulation under three different acidic conditions, namely 2 M acetic acid, 2 M hydrochloric acid, and 2 M sulfuric acid. The effect of coagulating medium on the final chemical composition of the hydrogel beads is investigated by spectroscopic techniques , and elemental analysis. The beads coagulated in 2 M acetic acid displayed an unchanged chitosan composition with free amino groups, while the beads coagulated in 2 M hydrochloric and sulfuric acid showed protonation of amino groups and ionic interaction with the counterions. The ultrastructural morphological study of lyophilized beads showed that increased chitosan content enhanced the porosity of the hydrogel beads. Furthermore, cytocompatibility evaluation of the hydrogel beads with human breast adenocarcinoma cells (soft tissue) showed that the beads coagulated in 2 M acetic acid are the most suitable for this type of cells in comparison to other coagulating systems. The acetic acid fabricated hydrogel beads also support osteoblast growth and adhesion over 192 h. Thus, in future, these hydrogel beads can be tested in the in vitro studies related to breast cancer and for bone regeneration. Polymers, derived from plant and animal sources, such as cellulose and chitosan, have functional groups that provide the unique characteristics, physicochemical properties, and are responsible for their cytocompatibility . ChitosaScaffolds prepared from various biomaterials have been used in hard (bone) and soft tissue (skin) engineering . The keyChitosan-based composite hydrogels, such as chitosan/alginate and chitosan/silica, have been tested to promote bone regeneration ,12. SimiN-methylmorpholine-N-oxide (NNMO), ionic liquids, and NaOH/urea/water solvent system are used to design chitosan-cellulose hydrogel beads of varying properties [In case of chitosan-cellulose-based hydrogel beads design, the solvent system plays a key role in the resulting properties such as chemical constitution, porosity, size, water retention capacity, which ultimately governs the application. Until now acetic acid, operties . Neverthoperties ,26,27.We aimed to design chitosan-cellulose hydrogel beads in an environment-friendly medium with variable ionic interactions of the protonated amino groups with the counter ions generated from the coagulating solvent systems. The cytocompatibility of novel materials to be used in biomedical applications is considered as the most crucial initial requirement for their potential application. Therefore, an effort was put to evaluate the same in our study. For this purpose, the novel hydrogel beads were tested towards human breast adenocarcinoma (MDA-MB-231) cells as an example of a soft tissue and towards human osteoblast cells as an example of hard tissue. Initially, we tested the basic cytocompatibility of the novel hydrogel beads, while based on the positive results, additional attachment testing was performed in the case of the osteoblast cells. Finally, a multi-day exposure of the cells to the developed materials was performed to evaluate their potential for tissue engineering applications.In a previous study, the NaOH/urea/water solvent system was used to prepare the chitosan-cellulose composite solution by freeze-thaw cycles at low temperatures . Thus, w2)2, 99.5% purity), sulfuric acid , acetic acid , and hydrochloric acid  were obtained from Merck. Ethanol (92%) was purchased from VWR. Deionized water was obtained from the Milli-Q system (0.2 µm filters). MDA-MB-231 cells (Human breast adenocarcinoma), Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 2 mM l-glutamine, and 1% penicillin, streptomycin (v/v) were purchased from Life Technologies, ThermoFisher Scientific Inc., Darmstadt Germany. WST-1 cell proliferation reagent was from Roche Diagnostics, Germany. 10% DMSO, Human bone osteoblasts (ATCC CRL-11372), DMEM with 5 wt.% FBS were purchased from Life Technologies, ThermoFisher Scientific Inc., Darmstadt, Germany and 3--2,5-diphenyltetrazolium bromide (MTT) assay was purchased from Sigma-Aldrich, Hamburg, Germany.Enoalfa cellulose pulp with alpha cellulose content >93.5% was provided by Enocell pulp mill, Finland. Low molecular weight chitosan (190–310 kDa with 75–85% degree of deacetylation) was purchased from Aldrich, sodium hydroxide  was purchased from Fluka. Urea . The pH value of coagulating medium was recorded after hydrogel beads gelation of the 70A, 70B, and 70C  samples. Finally, the hydrogel beads were stored in deionized water.To prepare chitosan–cellulose hydrogel beads, varying proportions of chitosan (10–90%) were mixed with HyCelSol  treated −1 resolution and 64 scans. Raman spectra were collected using a diode laser (power–0.5 W). The detector was InGaAs with CaF2 splitter, resolution 8.0 cm−1, aperture size 200, and number of scans 64,000. Lyophilized hydrogel beads were cross-sectioned and were analysed.Nicolet iS 50 FT-IR spectrometer with Raman module from Thermo Scientific  was used for spectrometric measurements. FT-IR spectra were collected using tungsten-halogen source and DLaTGS-KBr detector splitter setup with 4.00 cm13C and 15N NMR spectra were recorded with 400 MHz Bruker AVANCE-III NMR spectrometer, equipped with a 4 mm Cross-Polarisation Magic Angle Spinning CP-MAS probe operating frequencies 100.52 MHz (13C) and 40.51 MHz (15N). The spectra were recorded at the spinning rate of 5 kHz and a contact time of 2 ms (13C) and 3.5 ms (15N).The solid-state 3 using a chloride-sensitive electrode.A VARIO EL III CHNS analyser  was used for elemental analysis according to standardised procedures. The chlorine content was determined subsequently by combustion of the organic samples and potentiometric titration with AgNOα X-ray source and an HI-STAR area detector. The incident angle was kept at 6°, while the detector angle (2θ) was 25°.XRD measurements were performed on a Bruker D8 Discover instrument  with a Cu KThe qualitative composition analysis of beads was performed with JEOL JSM-6335F  with accelerating voltage 15 kV, Working Distance (WD) = 15 mm. Air dried cross-sectioned beads were moulded into the epoxy resin in a gelatinous capsule. The resin was allowed to cure at RT for 24 h. Trimming was performed with a microtome. After trimming, the sample was sputtered with platinum and a fresh surface exposed by slicing a thin layer off of the surface with the microtome. A spot analysis was taken with a COMPO image detector from two places representing different phases of the sample denoted as shades of lighter and darker grey.2.The cross-section analysis of beads was performed with LEO Gemini 1530. A Thermo Scientific Ultra Silicon Drift Detector (SDD) equipped with secondary electron backscattered electron and In lens, detector was used. The chitosan-cellulose hydrogel beads were frozen in liquid nitrogen and were lyophilized. The beads were further cross-sectioned, coated with carbon and analyzed. The magnification of the images corresponds to a Polaroid 545 print with the image size of 8.9 × 11.4 cm2 environment using MDA-MB-231 cells. MDA-MB-231 cells (Human breast adenocarcinoma) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10 wt.% fetal bovine serum (FBS), 2 mM l-glutamine, and 1% penicillin–streptomycin (v/v). 0A, 70A, 0B, 70B, and 0C, 70C, hydrogel beads that were previously soaked in cell culture medium were placed in a 96 well cell culture plate. MDA-MB-231 cells were added to each type of hydrogel beads for evaluation of attachment and proliferation for 48 h. After incubating the cells with hydrogel beads for 48 h at 37 °C, 5 wt.% CO2, 10 µL of WST-1 cell proliferation reagent  was added to the respective wells containing 100 µL of cell growth media, and the plate was again allowed to incubate for 3 h at 37 °C, 5 wt.% CO2. After the incubation period, the absorbance was read at 430 nm by Tecan Ultra microplate reader  from Bradenton, FL, USA. The number of viable cells was correlated with the observed absorbance from each sample. 10 wt.% DMSO (toxin) was added as a negative control, whereas wells without beads (tissue culture plastic (TCP)) were used as positive control cells and were allowed to grow without any cellulose beads.The cell studies were performed under standard conditions in a humidified, 37 °C, 5 wt.% CO0A, 50A, and 70A hydrogel bead samples suspended in ultrapure water obtained from Milli-Q were used for the osteoblast cells adhesion and proliferation studies. The number of hydrogel beads used was three 0A and two 50A and 70A respectively. The beads were sterilised using UV light (for 30 min) followed by soaking in 1 mL of the cell culture medium (advanced DMEM) with 5 wt.% FBS for 30 min. Human bone osteoblasts (ATCC CRL-11372), were seeded on a P96 well microtiter plate at a concentration of 20.000 cells/cm2. Allowing the cells to attach for 24 h, the hydrogel beads samples were added in four repetitions. For this purpose, we used the as-prepared samples, and their dilutions of 1:2, 1:4, and 1:8 in Advanced DMEM with 5 wt.% FBS. After one, four, six, and 192 h of incubation at 37 °C and 5 wt.% CO2, the sample cytocompatibility/cytotoxicity was assessed using the MTT assay . The media was changed every other day. Cell viability was determined via the reduction reaction of the tetrazolium salt MTT -2,5-diphenyltetrazolium bromide), as determined by measuring the absorbance  at 570 nm [The ight for  min foll0A, 50A, and 70A hydrogel bead samples were stored in ultrapure water obtained from Milli-Q. Four beads of sample 70A, and five beads of samples 0A and 50A were poured with 1 mL of Advanced DMEM medium with 5 wt.% FBS and left to culture with it for 30 min. Since the beads were of different sizes and had therefore different weights, as well as different surface to volume rations, our experiments were based on using same weights of the beads instead of the same number. Consequently, the number of beads for respective samples used for the experiments is different. The samples were transferred to a chamber with eight wells  and combined in the cell suspension with a cell density of 17,500 cells/well. The same cell density was used for the control well (as the control we used Advanced DMEM medium with 5 wt.% FBS without beads in tissue culture plastic plates (TCP)). Samples were prepared in duplicates. The obtained cells with beads were cultured at 37 °C, using an atmosphere of 5 wt.% CO2. The growth medium was replaced after 72 h cells seeding. After 192 h the samples were stained using the crystal violet dye . For this purpose, the medium was pipetted from respective wells, followed by washing of the samples twice with PBS , poured with 300 µL of the as-prepared dye solution and left for 5 min at room temperature. The dye was consequently pipetted away, and the samples were washed three times with ultrapure water obtained from Milli-Q system. This was followed by observing the surface of the beads with a Leica DM2500 microscope .The p < 0.05 (and lower) was considered as significant. All of the statistical tests were performed using Excel 2016.The data obtained for both the cell-based tests is presented as mean values ± standard deviation of the mean. Statistical analysis of the significance for the cytocompatibility testing on both cell types was performed using a one-way analysis of variance (ANOVA test) against respective control samples. A A, B, and C, respectively  solvent system. The beads were extruded in 2 M acetic acid, hydrochloric acid, and sulfuric acid, and named as ectively . The hydA) type, hydrochloric (B) type, and sulfuric acid (C) type, respectively shows an increasing trend in the nitrogen content, which corresponds to the chitosan content in hydrogel beads from initial 0 to 70% composition. The 70A has the highest nitrogen content equivalent to 3.4 mmol/g, while 70B has 1.2 mmol/g and 70C 2.9 mmol/g, respectively. The beads coagulated in 2 M hydrochloric acid had the lowest nitrogen content in comparison to the two other systems (3+) and had ionic interactions with the chloride (Cl−) and hydrogen sulphate (HSO4−) or sulphate (SO42−) ions. The qualitative analysis of the beads via SEM-EDS displayed shades of lighter and darker grey corresponding to different positions in the bead samples, which has been indicated by arrow in . It could be explained by the study by Cui et al., which showed that in the presence of sulfuric acid, the amino groups of chitosan are protonated (NH3+), but are in strong electrostatic interaction with the sulfonate (SO42−) anions [In the case of ) anions . Therefo−1, corresponding to O–H stretching and N–H vibrations. The presence of amide I band at 1651 cm−1 due to the carbonyl stretching vibrations, and a bifurcated band with peaks at 1590 cm−1 due to amide II, corresponding to N–H bending vibrations and at 1560 cm−1, due to free NH2 bending vibrations are present in chitosan. A slight variation in the CHx deformations region at 1425 and 1374 cm−1, C–O–C and C–O stretching vibration region at 1200–950 cm−1 are also apparent  overlaps with the water absorbance peak. Also, in the band corresponding to free amino groups clear variations are observed. In 70A, the band appears in the chitosan in the same region at 1555 cm−1, while in 70B, a band with lower intensity exhibiting a slight shift towards right at 1525 cm−1 is visible. In the case of 70C beads, a peak at 1532 cm−1 of equal intensity exhibiting a small shift towards the right, is present. The shift in peaks towards the right in the case of 70B and 70C is presumably related to the protonated amino (NH3+) groups bending vibrations having interaction with the counterions [−1 can also be observed  ions [70B and 70C hydrogel beads, while in 70A beads, no such evidence was observed.Similarly, in the region 3600–3100 cmellulose b. In 70A42− ions . The eff2−) ions . Hence, 2SO4 treated chitosan reference samples, as well as the lyophilized hydrogel beads 70A, 70B, and 70C, were analysed by solid-state CP-MAS 13C- and 15N NMR spectroscopy. The characteristic signals observed in the CP-MAS 13C NMR spectra of cellulose and chitosan  when compared to cellulose (73 ppm). Moreover, the signals corresponding to residual acetyl groups are observed in the CP-MAS 13C NMR spectrum of chitosan, i.e., the signals at 22 ppm (CH3) and 173 ppm (C=O), respectively. When chitosan was treated with sulfuric acid, the NH2-groups are converted to NH3+-groups and are ionically interacting with sulfonate ions. This resulted in broadening of all the signals observed in CP-MAS 13C NMR spectra and shift of C1- and C4-signals  and cellulose (22°) reflections in the beads is apparent. However, the relative peak intensities in the 10°–13° 2θ region can be used to distinguish the pattern of composite beads equivalent to chitosan or cellulose. The 70A sample shows a pattern closer to the pure chitosan pattern, while 70B and 70C have patterns closer to cellulose. These observations could be explained by the fact that in 70A, the chitosan has not undergone any changes during coagulation in 2 M acetic acid, while in 70B, lower content of chitosan is present due to dissolution and leaching out from the hydrogel beads during gelation. In 70C, the peak shapes resemble more the regenerated cellulose. This behaviour could be due to ionic interactions between chitosan and sulphuric acid. Therefore, the coagulating medium affects the crystallinity and the final composition of the beads, as shown in  over 48 h was evaluated using the WST-1 cell proliferation assay. A comparison between positive control and 0A, 70A, 0B, 70B, 0C, 70C was performed, as shown in , making them appropriate model cells for evaluating the potential of the developed materials for soft tissue engineering applications. For this purpose, we performed a cell viability testing. The cytocompatibility of the shown in . DMSO wa-cellulose hydrogel beads coagulated in acetic acid with human bone-derived osteoblast cells, as shown in . It is clear that the prepared beads affect the growth of the used cells, especially in comparison to the cell growth after each of the time intervals. After 24 h, we see a lower viability of the cells grown together with the bead groups, whereas already after 48 h, all of the prepared samples  show an increased viability, when compared to the control. Both samples that included chitosan (50A and 70A) also showed a higher viability than the beads prepared from pure cellulose. Between them, the difference was in the range of rerpoted error for respective samples. After 96 h the viability was comparable to the control sample, whereas the samples 50A and 70A again outperformed the control sample after 144 h. Since at this time period, the sample 50A showed the highest viability of all the samples, this might indicate that a modest number of free amino groups (from chitosan) has the most positive influence of the growth of osteoblast cells, whereas an even hugher number of free amino groups could potentially lead to a higher surface charge, which seems still suits the cells, but to a smaller extent. Regardless of the respective viabilities, these results definitely show that all of the tested  beads are suitable for growth of osteoblast cells at least up to 144 h. The results show a comparative cytocompatibility study between the control sample, i.e., Advanced DMEM, After 192 h, we observed a decrease in the viability of the cells exposed to the bead samples, when compared to the control. Examination of the optical micrographs of the cells after the mentioned time period (not shown) showed no negative effect of the beads that could be observed in comparison with the control (no changes in cell morphology or shape etc.). It seems that the lower viabilities might be related to the fact that the cells have overgrown the experimental environment of the well in the P96 plate. In comparison with the control, where we do not see this decrease in viability, this effect is mainly due to the faster growth of cells in the case of sample exposure, potentially leading to contact inhibition. Further studies are of course necessary to confirm the latter via other methods, yet this is out of the scope of the present study.A crystal violet dye viability/cytocompatibility assay was also performed to check the cytocompatibility of the hydrogel beads. The result is summarized in . FinallySince our intention was not only to assess if the proposed hydrogel beads degrade in the cells native environment, which could hinder cell growth (70A) showed higher chitosan retention with free amino groups. Whereas, in case of a 2 M hydrochloric acid (70B), low amount of chitosan was present and the amino groups are ionically interacting with Cl− ions. While in 2 M sulfuric acid (70C), although high chitosan was present, amino groups were involved in ionic interactions with HSO4−/SO42− ions. The cytocompatibility evaluation of the hydrogel bead groups with breast adenocarcinoma cell lines displayed 70A type has the maximum cytocompatibility in comparison to other groups. Similarly, in the case of beads coagulated in 2 M acetic acid, the osteoblast cells showed effective adhesion to the bead surface, as well as an increased viability, when compared to the positive control. These results are presumably related to the form of amino groups present in the hydrogel beads. Concretely, the chitosan-cellulose hydrogel beads exhibiting free amino groups, led to a higher cell viability, when compared to the prepared samples, where the amino groups were involved in ionic interactions with the anions as in the case of Cl− and HSO4−/SO42−. Herein, we propose the chemistry and the mechanism behind the hydrogel beads coagulation in different acidic media, cytocompatibility with model soft and hard tissue cell lines and their future potential use in the bone tissue engineering applications.Cytocompatible chitosan-cellulose hydrogel beads were prepared with varying chitosan content and chemistry along the amino group. The hydrogel beads coagulated in 2 M acetic acid (


With the advent of massively parallel sequencing, oncogenic viruses in tumours can now be detected in an unbiased and comprehensive manner. Additionally, new viruses or strains can be discovered based on sequence similarity with known viruses. Using this approach, the causative agent for Merkel cell carcinoma was identified. Subsequent studies using data from large collections of tumours have confirmed models built during decades of hypothesis-driven and low-throughput research, and a more detailed and comprehensive description of virus–tumour associations have emerged. Notably, large cohorts and high sequencing depth, in combination with newly developed bioinformatical techniques, have made it possible to rule out several suggested virus–tumour associations with a high degree of confidence. In this review we discuss possibilities, limitations and insights gained from using massively parallel sequencing to characterize tumours with viral content, with emphasis on detection of viral sequences and genomic integration events.This article is part of the themed issue ‘Human oncogenic viruses’. Animal in silico using bioinformatics, by first removing human sequences followed by unbiased detection of viral traces in the remaining data. Variants of this approach have since been used in many subsequent NGS-based studies. These have confirmed previously described virus–tumour associations and added several other insights, including rare associations, novel recurrent sites of virus integration, and rejection of previously proposed associations. Here we review some of the contributions NGS has made in this field, with the main focus on detection of viral sequences in cancer.The most recently discovered human tumour virus, Merkel cell polyomavirus (MCV), responsible for the majority of Merkel cell carcinomas, was identified using a pioneering bioinformatical method, digital transcriptome subtraction of sequences generated by NGS . The pri2.NGS-based characterization of viral sequences in tumour material generally presents few experimental challenges, since standard protocols for nucleic acid extraction and sequencing are applicable. Sequencing libraries generated for general transcriptomic or genomic analyses of tumours can therefore be repurposed for viral analyses, which has enabled large cancer cohorts to be screened for viral content solely using bioinformatics and publicly available sequencing data –9.a).Typically, tumour tissue is flash frozen after harvesting, and pathological tissue slides are prepared and analysed to define the borders of the tumour. Samples with high tumour content are then lysed and nucleic acids are purified. The type of nucleic acid analyte analysed  will determine what types of viruses can be detected and what kinds of studies may be performed. For example, DNA-based analyses can reveal integrated and latent non-expressed viruses, and may enable quantification of absolute viral load per human cell . Transcrfigure 1a) [b) [figure 1c, discussed below). Several software packages are now available to simplify these tasks, reducing the expertise required [The bioinformatical analysis generally involves removal of low-fidelity reads followed by matching against human reference sequences. Remaining non-human reads are finally matched against a viral genome database a [7–9,11a) . Variabia) [b) ,15,16,18required ,17,18.3.Early NGS-based studies of tumour viruses were limited by the relatively low sequencing depth available at the time. Bioinformatical processing was carried out meticulously with every sequence read analysed and categorized . TranscrAnalysis of 85 cervical squamous cell carcinomas and endocervical adenocarcinomas, and 43 head and neck squamous cell carcinomas, infected with various types of high-risk human papillomaviruses (HPV), revealed an average of approximately 200 ppm of viral mRNA reads in relation to the total library size, ranging from 11 to 598 ppm for cervical squamous cell carcinoma and endocervical adenocarcinoma, and 22 to 848 ppm for head and neck squamous cell carcinoma. Similarly, the average hepatitis B virus (HBV) mRNA content in 11 liver hepatocellular carcinomas was also nearly 200 ppm, ranging from 2 to 854 ppm with three tumours containing less than 10 ppm viral mRNA [Results from six AIDS-associated lymphomas containing Epstein–Barr virus (EBV/HHV4) revealed viral expression ranging from 145 to 8857 ppm, with an average of 2750 ppm EBV transcripts . InteresA limit of 10 ppm viral mRNA reads  has been suggested as an approximate divider for tumours with clonal presence of expressed viruses, since most virally induced tumours were found to surpass this level . Lower lRecent studies employing small RNA sequencing, whole exome sequencing (WXS) and WGS data for identification of viruses in tumours have produced results that are largely consistent with transcriptomic analyses ,21–23,254.in situ hybridization to be due to infiltration of latently EBV-infected lymphocytes [The sensitivity and unbiased nature of NGS gives rise to a new type of problem, where trace amounts of human as well as non-human viruses are often detected in tumours and control tissues –28. Thesphocytes . Beta  infection occurs predominantly in the oropharyngeal area with ensuing cold sores . PossiblAn additional challenge is contaminants, which may be introduced at all steps during sample preparation or downstream processing . Silica Sequences from non-human viruses are typically present only in low amounts when detected by NGS in tumours ,26. AlthA frequently found synthetic viral contaminant is the immediate early promoter of the human cytomegalovirus (HHV5 or CMV) used in many mammalian expression plasmids –11,25. Ain situ hybridization, immunohistochemistry and PCR of tumour and healthy tissue are useful to confirm initial findings [The examples described here stress the need to maintain a critical standpoint towards novel virus–tumour associations detected by NGS. Negative control samples and complementary laboratory assays such as findings ,21,24,41findings ,42. Finafindings .5.Viruses that have co-evolved with humans as their main host are typically highly selective in terms of the cell types they can infect . Detectiin situ hybridization or immunohistochemistry is needed to confirm these observations.HPV16 is one of the few viruses that have been associated with tumours outside the primary sites of infection in the ano-genital region. In addition to expected detections in head and neck and cervical tumours, HPV16 has been found in single uterus, lung and bladder carcinomas sequenced by TCGA ,9,11. WhEBV is another agent implicated in a wide range of cancers including Burkitt's lymphoma, nasopharyngeal carcinoma, Hodgkin's lymphoma and gastric adenocarcinoma. Additionally, transcriptomic analysis recently revealed high levels of EBV in 2 out of 105 diffuse large B-cell lymphomas (432 and 37 ppm) . Two tumA single bladder urothelial carcinoma, out of 316 characterized using transcriptome sequencing by TCGA, was found to contain BK polyomavirus (BKV) ,9,20. Ea6.in situ hybridization and western blotting, all of which are prone to false positive detections. Modern genomic approaches, which allow unbiased screening of large tumour cohorts, have the potential to bring clarity to some of these proposed associations.The literature contains a large number of proposed virus–tumour associations that are controversial. These are typically based on traditional viral diagnostic techniques, including PCR, immunohistochemistry, de facto causative agent [During the 1960s and 70s, it was widely believed that HSV2, which causes genital herpes, was the causative agent for cervical carcinoma. High-risk HPV types were later identified in these tumours and years of disputes followed  before hve agent . Today, ve agent ,23. HencBreast cancer is the most common invasive cancer in the world, and has been extensively studied. Several viruses including EBV, HPV and MMTV (mouse mammary tumour virus) have been implicated . FrequenThe role of CMV in human cancer has been highly controversial. After the initial claim that CMV DNA and protein was found clonally in the majority of gliomas , CMV has7.Retroviruses such as HTLV-1 (human T-lymphotropic virus type 1) establish chronic infection by genome integration, which causes the virus to be propagated in the host for long periods of time ,58. Intec) [a), and by combining transcriptome and WGS data [Two main principles, applicable to both DNA and RNA sequencing data, are used for detection of viral integrations c [7–9,11c) ,40,63–65WGS data ,66.HPV integrations have been studied for decades, and methods for enrichment and sequencing of integration points have been developed both for fusion transcripts and integrated DNA . Recent FN1 gene were frequent only in non-cancerous tissue [Likewise, HBV integration has been thoroughly studied, and recurrent viral integrations have been detected in specific genomic regions . During s tissue ,69–71.EBV integration has previously been described in cell lines, but not in tumours. Surprisingly, transcriptomic analysis of 24 EBV-positive gastric adenocarcinomas revealed several reads that supported integration in one single tumour, suggesting that EBV integration may occur also in clinical samples .Comprehensive mapping of HTLV-1 integrations by enrichment of insertion sites before sequencing has enabled monitoring of latently infected CD4+ T-lymphocyte populations before the onset of adult T-cell leukaemia, and has revealed that malignant cells most often originate from single clones and not through oligoclonal proliferation as previously proposed ,65. As e8.in silico methods are highly dependent on existing databases of viral sequences, and many new viruses, including potential human pathogens [NGS was successfully used in the discovery of MCV in Merkel cell carcinoma , but it athogens ,73, haveSequencing technologies continue to improve at a rapid pace. Each new generation of machines promises longer reads, shorter run times, and lower per-base-pair cost. While current technology is more than sufficient for the detection of clonally present viruses in tumours, many benefits can still be expected as the amount of data continues to increase. For example, more widespread use of total RNA transcriptomic analyses, rather than the polyA enriched protocols that dominate today, will provide a more comprehensive description of cellular RNA content including non-polyA viruses. High-coverage WGS, which is becoming increasingly accessible, gives improved possibilities for mapping of viral integration breakpoints, quantifying viral load, and better statistical strength for rejecting controversial virus–tumour associations. Larger cohorts will increase the chances of finding new viruses or rare associations. Furthermore, single-cell sequencing approaches will be able to shed light on heterogeneity in cell populations, including the interplay between virus-infected and surrounding cells .The discovery of human tumour viruses has had a profound effect on the prevention of human cancer. Efficient vaccines and antiviral treatment have completely shifted the aetiological causes for cancer in developed countries. It is therefore of great importance for the scientific community to remain vigilant in the search for new virus–tumour associations. NGS is now proven to be extremely efficient for characterization of tumour viral content, and will soon be the primary tool for discovering, confirming, as well as rejecting virus–tumour associations.


Comparisons of immature and mature virions show large rearrangements in the glycoproteins that may be essential for forming the infectious virions. These results present the first known example of a helical membrane-enveloped virus, while also providing a structural basis for its assembly and maturation pathway.Viral infections during pregnancy are a significant cause of infant morbidity and mortality. Of these, rubella virus infection is a well-substantiated example that leads to miscarriages or severe fetal defects. However, structural information about the rubella virus has been lacking due to the pleomorphic nature of the virions. Here we report a helical structure of rubella virions using cryo-electron tomography. Sub-tomogram averaging of the surface spikes established the relative positions of the viral glycoproteins, which differed from the earlier icosahedral models of the virus. Tomographic analyses of  In vitro assembly studies on the virus capsid protein have provided insights into the interactions necessary for virus assembly. Comparisons between mature and immature RV show large conformational changes in the virion structure that are essential for virus maturation. These results help to gain a structural understanding of RV pathogenicity, which may also be relevant to other teratogenic viruses.Rubella virus (RV) causes serious fetal defects when contracted during pregnancy. Despite its medical importance, due to the irregular shapes and different sizes of the virions, the RV structure has remained unknown. Using cryo-electron tomography, we have determined the RV structure, which shows a unique, helical outer surface. Subsequent local averaging of the RV surface spikes has established the conformations of its immunogenic glycoproteins.  Rubella virus is an airborne human pathogen that causes a contagious disease with measles-like symptoms in children and adults. Rubella infection in pregnant women can lead to fetal death or severe life-long disabilities such as mental retardation, deafness, cataracts and heart defects in the new-born infants, collectively referred to as congenital rubella syndrome . DespiteRubella virus is an enveloped, positive-stranded RNA virus with virions ranging from 500 Å to 900 Å in diameter . The virThe rubella virus structural proteins assemble on and bud from Golgi membranes , 4, 17 iBased on their similar genome organizations, rubella virus and alphaviruses are classified as the only members of the Togavirus family , 19, 20.in vitro assembly studies to elucidate the three-dimensional structure and assembly of rubella virions. We have further taken advantage of the direct detector technology to achieve close to nanometer resolution for the sub-tomogram average of the asymmetric and relatively small rubella virus glycoprotein spikes (~100kDa molecular weight). Additionally, we have isolated immature rubella virions that give insights into the different maturation states of rubella virus. Collectively, these analyses suggest a distinctive assembly and maturation pathway for rubella virus that might be critical for the teratogenic pathogenicity of this medically significant virus.In this report, we have used a combination of cryo-electron tomography, sub-tomogram averaging and Samples of mature rubella virus were prepared for cryo-electron tomography as described previously . About 3Variable arrays of surface glycoprotein “spikes” project outwards from the rubella virus membrane , 16 Fig. HoweverThe rubella glycoproteins (E1 and E2) are present as heterodimeric complexes or spikes on the surface of the virus , 8. Howe3. From this, the volume occupied by the fitted E1 ectodomain (PDB ID: 4ADG) was subtracted, leaving a residual volume of 52 Å3 at the base of the spike. The residual volume can accommodate a protein of roughly 33kDa molecular weight. This is larger than the molecular mass of the E2 ectodomain polypeptide (25kDa) but is smaller than the estimated mass of the glycosylated E2 ectodomain (37kDa to 42kDa). Taking into consideration that the glycosylation on the E2 ectodomain is known to be heterogeneous [The volume of the averaged spike was 133 Åogeneous , it is lThe arrangement of glycoprotein spikes on rubella virions was further examined by placing the sub-tomogram averaged spike, back into each of the original tomographic positions that had been used to obtain the averaged spike structure. The relative orientations of the long axes of the glycoprotein spikes to the viral membrane varied from 30° to 90°. Along glycoprotein rows, adjacent spikes were slightly rotated with respect to each other, along an axis perpendicular to the plane of the membrane. This analysis showed that along unbroken glycoprotein rows in rubella virions, the glycoprotein complexes are similarly oriented with respect to each other, such that E2 would most likely be located between adjacent E1 positions .The internal nucleocapsid shell of rubella virus consists of the capsid protein and the viral RNA genome. The nucleocapsid surface follows the contour of the viral membrane . In previn vitro to form nucleocapsid cores . These in vitro assembled rubella virus nucleocapsid cores have a smooth exterior and are hollow. They display a variety of shapes and sizes with diameters ranging from 400 Å to 900 Å . Tomogram sections of the nucleocapsid cores show a tetramer-like pattern as seen in the assembly intermediates . This showed that each monomer of the tetrameric unit has a dumbbell-shaped structure  . The N-t program  . Cryo-electron microscopy of the purified, immature virus particles shows that the immature virions are uniformly dense and variable in size  corroborThe rubella virus structure had been expected to have T = 3, icosahedral symmetry , 8, 41. The relative positions of the rubella glycoproteins, with an extended E1 conformation and with E2 at the base of the spike complex, is different from the glycoprotein heterodimer conformation observed in alphaviruses. However, this structural placement of rubella glycoproteins is consistent with protease studies , 45 and In alphaviruses, the glycoprotein E1-E2 heterodimers lie close to the membrane surface in their pre-fusion state with the E1 fusion loop physically masked by the neighboring E2 molecule. In the rubella glycoprotein complex, the E1 protein extends out from the virion surface and does not appear to require E2 for shielding its two fusion loops. The rubella virus E1 crystal structure , which wDifference in the glycoprotein spike orientations between the immature and mature virion forms could also be to protect the fusion loops on the E1 glycoprotein structure from undergoing unproductive membrane interactions in the low pH environment of the cellular Golgi network. Subsequent virion maturation in the Golgi complex would then be necessary for reorganization of the viral envelope to yield fusion competent, extracellular, ‘spiky’ mature virus particles. A similar strategy of surface glycoprotein reorganization during virion maturation occurs also in bunyaviruses  and flavIn addition to the glycoprotein arrangement, the association of protein tetramers to form enclosed shells as seen in the rubella virus nucleocapsids is also unusual and not observed in other known virus structures. Rubella capsid protein molecules expressed in bacterial cells, spontaneously form nucleocapsid cores in the presence of cellular nucleic acids. This indicates that the rubella capsid protein does not need the rubella genome for nucleocapsid formation, similar to previous observations , but canThus, rubella virus appears to share certain common characteristics, not only with alphaviruses but also with other arbovirus genera, such as flaviviruses and bunyaviruses. However, in addition to these features that suggest similarities to arboviruses, rubella virus has also evolved some unique traits such as an exposed E1 fusion loop conformation. Together, these observations point to a more complex structural evolution in rubella virus than previously assumed.2 and then homogenizing the suspension in a Dounce homogenizer. The cell lysates were centrifuged at 3000×g for 5min to pellet cell debris. The supernatant was further centrifuged at 20,000×g for 20 min. The supernatant was again collected and incubated with 5mM EDTA for 10min followed by addition of 10μg/ml of RNase A. After 15 minutes, the supernatant buffer concentration was adjusted to contain 120mM NaCl. The supernatant containing the immature virions was further purified similar to the mature virus purification protocol [Mature rubella virus M33 strain) was cultured in Vero cells (ATCC CL-81) and purified according to previously published protocols 3 strain  was follE. coli BL21 (DE3) cells (Novagen). Cells were grown at 37°C and induced with isopropyl thiogalactoside (IPTG) when the O.D reached 0.8. The induced cultures were grown at 25°C for 5 hours and pelleted. Cell pellets were re-suspended in buffer containing 50mM Tris (pH 7.2) and 500mM NaCl. The cells were lysed using sonication and the target protein purified using a chitin column (New England Biolabs). The purified RVC-9-277 protein was applied to a Sepahcryl S-400 column  to fractionate the sample according to different apparent molecular weights. Fractions corresponding to assembled nucleocapsids and smaller capsid protein complexes were pooled and concentrated separately. The nucleocapsid core samples were treated with 0.001% benzonase and the buffer was exchanged before sample preparation for cryo-electron tomography.The rubella virus capsid gene has two methionines in the first 10 nucleotides of its sequence (at positions 1 and 9). To improve bacterial expression, the nucleotide sequence corresponding to amino acids 1 to 8 was removed. Rubella virus (M33 strain) capsid protein (RVC) gene sequence corresponding to amino acids 9–277 was cloned into a pTXB1 plasmid (New England Biolabs). The RVC-9-277-pTXB1 construct was transformed into For cryo-electron tomography, the purified samples were mixed with 4X concentrated 10nm BSA gold solution  in a 15:1 volume ratio. 3μl of this mixture was applied to Quantifoil R1.2/1.3 grids (Electron Microscopy Sciences), blotted and plunge frozen in liquid ethane.in vitro assembled nucleocapsid core samples. The total dose applied to each sample was between 80–90 e-/Å2 [Tilt-series data was collected using a Titan Krios (operated at 300kV) and a Gatan K2 Summit detector. The Leginon software , 35 was 90 e-/Å2 , 35.The tilt-series were aligned using gold fiducial markers and reconstructed using the IMOD software . CtfplotFor negative staining, 1% solution of ammonium molybdate was added to the sample on a grid, blotted, washed twice with distilled water and then air dried after blotting off the excess liquid. The grids were analyzed using a CM200 (FEI) microscope with a 1k×1k CCD camera.Sub-volumes of virions were extracted from the cryo-electron tomograms using IMOD . The repFor sub-tomogram averaging of individual glycoprotein spikes that were elongated and predominantly straight, 7290 sub-volume positions were picked on the surface of virions in all directions from 8X binned tomogram data using IMOD . Each glAs the spikes on the virion surface are densely packed and touch each other at the base, a tight cylindrical mask with a radius of 6 pixels (31.6 Å) and a soft Gaussian fall-off of 6 pixels was applied to the particles such that only one spike was visible in the extracted sub-volumes. Before starting the alignment and averaging procedure, all the particles were rotated to align the long axis of the spikes (given by the two identification points) in one common direction. Individual particles were checked to make sure that all the spikes were oriented in the same direction with no inversion of the spikes’ orientations. The search parameters allowed for a complete 360° search about the long axis of the spikes, but restricted the search in the other two directions to ±30° with 0° being along the long direction of the spikes. The initial alignment iterations were coarse searches followed by finer interval searches in later iterations. Missing wedge compensation feature within the PEET software , 38 was rd of all the spikes. Sub-volumes were aligned to the updated reference model at each iteration. After the final iteration, the determined relative position and angles for each sub-volume was used to average all the sub-tomograms together to give an averaged 4X binned map of the glycoprotein spike. The determined relative orientations and positions were then applied to 2X binned data and the final sub-tomogram average map was calculated using 2X binned data with a pixel size of 2.64 Å. The reference models at every alternate iteration along with the final averaged map are shown in Initially, five spikes that looked properly formed from different tomograms were selected and independently used to align 256 sub-volumes to calculate initial models. The model which best resembled the individual spike conformations was selected for further steps. The remaining sub-volumes were split into two halves and processed independently. The initial model was used as the reference model for aligning the individual spikes at the first iteration. For subsequent iterations, the reference model was updated by averaging spikes with the highest correlation coefficients to the reference model in the previous iteration, representing 2/32 to increase the impact of higher resolution terms in the map. This sharpened map was used for fitting of the E1 crystal structure.The final 2X binned averages from the two half-sets were applied with a soft-edged mask that covered only the glycoprotein spike in order to exclude the surrounding, weak membrane densities. A Fourier-Shell Correlation (FSC) curve was subsequently calculated between the two masked averages using the EMAN2 software suite . The subFor sub-tomogram averaging of rows of glycoproteins , 240 rows containing four spikes each, with approximately similar spacing between the spikes, were picked using IMOD  from 8X Sub-tomogram averaging of the recombinantly produced capsid tetramers were also performed in a similar way to the above described routine for glycoprotein rows. Twenty tetramer sub-volumes were picked from 8X binned tomograms of nucleocapsid cores. Alignment and averaging was performed using 4X binned data.S1 Table(DOCX)Click here for additional data file.S1 FigThe atomic model of the rubella E1 glycoprotein (red) is placed into one of the subunits of the averaged density to show that the averaged volume of the rubella glycoprotein rows only covers about half the volume of the E1 structure. The left and right panels are rotated 90° with respect to each other. Scale bar is 50 Å long.(TIF)Click here for additional data file.S2 Figrd of the total sub-volumes for iterations 3, 5 and 7. The bottom middle panel shows the raw final average derived for the 4X binned data. The bottom right panel shows the raw final average for 2X binned data. The black arrows indicate the positions of the top and bottom of the membrane region. Scale bar in black indicates 25 Å.From top left to bottom right: The top left panel shows the initial model derived from 256 initial spikes. The next three panels show the updated reference models derived from 2/3(TIF)Click here for additional data file.S3 FigFourier Shell Correlation (FSC) curve (gold-standard) between the masked averages from two independent half-sets of the glycoprotein spikes. The dashed line at 0.143 FSC intersects the curve at 11.0 Å.(TIF)Click here for additional data file.S4 Fig(A and B) The virions represented here are the same as in (TIF)Click here for additional data file.S5 FigLeft panel is an agarose gel under UV illumination to show the presence of nucleic acids. The right panel shows the same gel after Coomassie blue staining to indicate presence of protein. Lane 1: 1kb DNA ladder, Lanes 2 and 4: purified nucleocapsid cores, Lanes 3 and 5: purified nucleocapsid cores after benzonase treatment. Capsid protein in lane 4 (in the presence of nucleic acid) appears as a diffused band whereas in lane 5 the protein band is discrete. Black brackets indicate the protein regions in the stained gel.(TIF)Click here for additional data file.S6 FigLeft and right panel show the two best fit orientations of the capsid protein structure into one lobe of the capsid unit density.(TIF)Click here for additional data file.S1 Movie(MP4)Click here for additional data file.S2 Movie(MP4)Click here for additional data file.S3 Movie(MP4)Click here for additional data file.


Phlebovirus, family Phenuiviridae) which shows a wide distribution in the Mediterranean basin  is a sandfly borne virus  of a patient presenting with meningitis . Serolog2-CDC traps  with homologous sequences of other TOSV strains and selected phleboviruses belonging to the Bank see  12). Am. AmSandfThe two TOSV sequences were clearly different from each other with 3.8 and 16.5% genetic divergence at amino acid and nucleotide level, respectively. C63 sequence was grouped with the unique Croatian TOSV sequence (corresponding to the CSF sample of a patient presenting with TOSV meningitis in 2008) ; hence, b and cytochrome c oxidase subunit I as previously described  , 17. Theou virus , Balkan ou virus , and TOSou virus .With the assumption that only one insect in each pool is infected, the infection rate of TOSV in Croatia was 0.1% which is higher than rates observed in Tunisia (0.03%), Spain (0.05%), and Algeria (0.004%) , 21.Ct value 35.3) only the SFNV nested PCR was positive and resulted in a 320-nt long sequence. However, the length of the sequence did not affect neither the quality of the alignment nor the topology of the phylogenetic tree.Despite the efforts, virus isolation was not obtained probably because of virion degradation during field collection. Due to the low viral load in pool C64  Croatia is, after France and Turkey, the third country where two lineages of TOSV are sympatric; (iv) physicians should consider testing patients who present with neuroinvasive infections in Croatia during the season of sand fly activity for TOSV.In conclusion, this study showed that (i) strains of TOSV lineage B are present in coastal Croatia where they cocirculate with lineage C strains; (ii) Sequences generated in this study are available in the GenBank database under the accession numbers KY867756 and KY867757.NA performed the sample collection, administered experiments, and wrote the manuscript. BA, VI, FM, OK, and YO organized the sample collection and contributed to the manuscript. XL designed the experiments and contributed to the manuscript. RC designed the experiments and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Recent studies have established that the human urine contains a complex microbiome, including a virome about which little is known. Following immunosuppression in kidney transplant patients, BK polyomavirus (BKV) has been shown to induce nephropathy (BKVN), decreasing graft survival. In this study we investigated the urine virome profile of BKV+ and BKV− kidney transplant recipients. Virus-like particles were stained to confirm the presence of VLP in the urine samples. Metagenomic DNA was purified, and the virome profile was analyzed using metagenomic shotgun sequencing. While the BK virus was predominant in the BKV+ group, it was also found in the BKV− group patients. Additional viruses were also detected in all patients, notably including JC virus (JCV) and Torque teno virus (TTV) and interestingly, we detected multiple subtypes of the BKV, JCV and TTV. Analysis of the BKV subtypes showed that nucleotide polymorphisms were detected in the VP1, VP2 and Large T Antigen proteins, suggesting potential functional effects for enhanced pathogenicity. Our results demonstrate a complex urinary virome in kidney transplant patients with multiple viruses with several distinct subtypes warranting further analysis of virus subtypes in immunosuppressed hosts. Prominent examples in the latter category include BK virus (BKV) and JC virus (JCV), both members of the Polyomaviridae family. Primary infection with BKV and JCV occurs in healthy individuals, often during childhood, and is detected by sero-positivity of anti-viral antibodies. Following infection, these viruses commonly becomes latent; however, they persist in the urinary tract and can reactivate in the context of immunosuppression following transplantation3567Kidney transplantation is the preferred treatment modality for most patients with end stage renal disease (ESRD) and offers an enhanced lifestyle and longer survival for many patient groupsThe majority of BKV and JCV infections are diagnosed by quantitative PCR in the urine and blood or by use of decoy cells (cytology)610While the virome has been less studied than its bacterial counterpart in human microbiome analyses14162021p < 0.001, Anelloviridae, Adenoviridae, Herpesviridae, Papillomaviridae and small numbers of unclassified viruses . We found multiple virus subtypes in several individual patients. In an analysis of BKV subtypes, we mapped the 25 subtypes identified in our sequence data onto the 81 known BKV reference genomesThe BK virus family contains subtypes specific for animal species ranging from mice to chimpanzees. In a hierarchical clustering analysis of 26 species-specific BKV subtypes -linked sialic acid-containing gangliosides GT1b and GD1b. Mapping VP1 sequences onto the phylogenetic tree of reference BK subtypes  gives a We also observed polymorphisms in amino acids of the VP2 protein as predicted from Dunlop (1a) and the BKV genomes of B9/RU13 (IV) and B12/A-62H (1a), using ClustalW. The VP2 protein is a multifunctional structural protein in the BKV capsid and is involved in the binding to the host cell receptor together with VP1 protein. Overall, the predicted protein sequences were highly similar . In anotLarge T antigen (LTA) is a DNA binding protein of polyoma viruses that regulates immediate early gene expression following infectionAnelloviridae family viruses in multiple patients with the bulk being TTV (genus Alphatorquevirus). Ten patients had unclassified Anelloviridae, which was frequently abundant, but no patient had detectable Gammatorquevirus. For example, 87.9% of the total virus abundance in patient B16 was unclassified Anelloviridae. A recent study in lung transplant population noted a more complex population of Anelloviruses in broncho-alveolar lavage samples as compared to healthy controlset al., noted increased abundance of the Anelloviridae family upon immunosuppression, a trend also exhibited by polyoma viruses in immunosuppressed patientsThe development or reactivation of viral infections in immunosuppressed patients (such as transplant patients) causes significant morbidity and mortality. BKV has been linked to renal disease in organ transplant, and JCV has been associated with renal tubular epithelial infection and progressive multifocal leukoencephalopathy (PML). In this study, we used metagenome shotgun sequencing rather than the 16S rRNA amplicon based sequencing approach, as the latter is limited to the identification of bacteria classified by operational taxonomic units (OTUs) which impute genera. In contrast, the shotgun metagenomics strategy enables sequencing of nearly complete genomes and identifies all components of the microbiome including viruses, and even allows identification of virus subtypes and polymorphisms in specific amino acids in major virus proteins. Though the number of reads generated by the shotgun sequencing methods are generally not even across the samples and could potentially be biased due to an unequal number of sequences. However, the normalization procedures makes the relative abundance counts/ratios comparable among different samples. Previously, we have shown that in shotgun sequencing data a similar microbiome profile is detected in each sample irrespective of sampling depth in the same sample31Adenoviridae that cause 2–5% of human respiratory infections, gastroenteritis and other human infections. Sapovirus includes the Sapporo virus, one of the two most common causes of acute viral gastroenteritis in adults. Whether urinary virus correlates with an acute infection remains to be determined. The Papillomaviruses were predominantly Betapapillomaviruses, with Alphapapillomavirus or Gammapapillomavirus, each only detected in one patient. The “unclassified” viruses match sequences in the database that have not been classified as a subtype. Lentivirus, a genus of the Retroviridae family that includes HIV and Visna virus, was present at 8.3% in a single patient. Similarly, Totivirus, which has fungi as natural hosts, was also detected at >8% in a single patient (B25) that, interestingly, also had detectable levels of Candida tropicalis and Malassezia globose (data not shown). There were also 11 patients with unclassified viruses which ranged in abundance up to 41.7% relative to other viruses, suggesting that the transplant population is infected with additional viruses that are not yet classified or well understood , Sapovirus (positive in 4 patients) and Papillomavirus (positive in 5 patients). The genus Mastadenovirus encompasses 25 species including derstood . Of noteA striking result in our study was the observation of multiple subtypes of the major urinary viruses BKV, JCV and TTV. For example, in total we detected 58, 31 and 108 different types of BKV, JCV, and TTV respectively, across the BKV+ and BKV− groups . Interes212240Taken together, our results indicate that viruses were detected in all of our study patients, whether or not they were classified as BKV+ or BKV−. Also, many patients did not have a single dominant subtype, but rather had multiple virus subtypes. It will be important to determine the pathogenicity of the various virus subtypes in immunosuppressed patients.The objective of this study was to investigate the urinary virome in kidney transplant recipients with and without BK polyomavirus infection, using metagenome shotgun sequencing. The urine samples from the kidney transplant recipients with known comorbidities were analyzed. All Adult kidney transplant patients who received kidney transplants at the Washington University Medical Center, St. Louis, MO, were eligible for inclusion in the study. All patients were treated with a standard of care protocol. Four doses of rabbit anti-thymocyte globulin  were administered; the first intra-operatively followed by doses on postoperative days 1–3. Dose adjustments were made for leukopenia or thrombocytopenia. Calcineurin inhibitors were instituted upon a brisk diuresis but no later than 4 days post-operatively. Cyclosporine was started at 8 mg/kg per day in two divided doses targeting 12 h whole blood trough levels of 250–300 ng/mL, which was monitored using a monoclonal fluorescent polarization immunoassay (FPIA)  during the first 3 months, then adjusted to 200–250 ng/mL. Alternatively, FK506 was initiated at 0.1 mg/kg per day divided in two doses adjusted to trough levels of 5–10 ng/mL by microparticle enzyme immunoassay (MEIA) . Methylprednisolone (7 mg/kg IV) was infused intra-operatively, then prednisone was started at 1 mg/kg per day, orally, postoperatively and tapered to 5–7.5 mg daily by the third month. All patients received prophylactic antibiotics (Bactrim). All urine samples were collected within the first year following transplantation during routine standard of care clinical follow-up. Clinical information including laboratory results, infections, and medications were collected for each subject at the time of each clinic visit. Comorbidities including Type 1 Diabetes Mellitus (T1DM), Type 2 Diabetes Mellitus (T2DM), hypertension (HTN), focal segmental glomerulosclerosis (FSGS), post-streptococcal glomerulonephritis, nephrocalcinosis (PSGN), polycystic kidney disease (PKD), reflux nephritis (RN), systemic lupus erythematous (lupus) and vasculitis were documented. The demographics of the transplant population are described in g for 15 min at 4 °C to pellet and remove the mammalian cells, then the supernatant was centrifuged at 100,000 × g for 2 h at 4 °C in an ultra-centrifuge  to pellet the VLP and the microbial content, frozen at −80 °C. For the epifluorescence staining of the VLP, the pellet was re-suspended in 1 ml of 1 × PBS. stained with SyBr Gold  for 10 min in the dark and visualized by epifluorescence microscopy as previously described2 and subjected to DNaseI treatment (2.5 U/ml) (Invitrogen) for 15 mins at 37 °C, and the reaction was stopped by adding 2 mM EDTA, and heating the contents for 10 min at 65 °C. The contents were subjected to enzymatic treatment by adding 800 μl AL Buffer (DNAeasy kit (Qiagen), Lysozyme (Fisher Bio Reagents) and enzyme solution  for 2h at 37 °C. Proteinase K (80 μl)  was added to the mixture and the DNA was eluted using elution columns following the DNAeasy kit (Qiagen) protocol. The purified DNA was quantified using a Qubit fluorometer (Life Technologies Corporation), and equal amount of DNA from all samples were mechanically fragmented using a Covaris S220 (Covaris Inc). The DNA libraries for sequencing were prepared using the NEBNext DNA library prep kit for Illumina (New England BioLabs Inc). The quality and quantity of all the DNA libraries were analyzed with a Bioanalyzer 2100 (Agilent Technologies) and Qubit fluorometer. Equal amount of all the DNA libraries were sequenced on Illumina HiSeq 2000 generating 50 base single end reads (BGI Americas). Control libraries were prepared by using 1 × PBS buffer instead of urine, and were subjected to the same DNA isolation protocol as for test urine samples. As expected there was no detectable DNA by Qubit analysis. The control DNA was then used to prepare metagenomic libraries, but no DNA was detected by Bioanalyzer analysis and thus not quantifiable. The control libraries were pooled and sequenced with the test libraries. After de-multiplexing of the sequence reads, no reads were assigned to the control libraries.Urine obtained by mid-stream clean catch was processed in sterile hoods in a positive pressure airflow room. Urine was centrifuged at 180 × p < 0.05 unless otherwise indicated.DNA sequence reads were quality trimmed and mapped against the reference human genome to remove human DNA sequences using the CLC Genomics Workbench with the default parameters . To investigate the urinary virome profile we used two approaches. First, the filtered non-human reads were analyzed using BLAST against the NCBI-nt database to ascertain the microbial taxonomy profile for each sample. The domain level taxonomy profile for bacteria, virus, eukaryotes and unclassified sequences was generated and relative abundance was calculated. From these total reads, the virus only reads were selected in-silico based on blast based taxonomy profile of each sample. All the reads identified from virus domain were selected in Fasta format and the respective taxonomy in csv format files. Second, to further investigate the nucleotide substitutions in whole genome and to determine the structure of major encoded proteins, the filtered non-human reads were assembled into contigs using CLC Genomics Workbench using default parameters. The contigs annotated to virus were identified using NBCI-nt BLAST. Virus counts for each sample were normalized using the RPKM (Reads per kb per million reads) and has been described in 1926Phylogenetic tree analysis was based on the complete genome sequences of the reference BK, JC and TT virus genomes and partial genome sequences of isolates identified in BKV+ and BKV− kidney transplant samples. The evolutionary history was inferred by the Maximum Likelihood method based on the Tamura-Nei model. The bootstrap consensus tree inferred from 100 replicates was assumed to represent the evolutionary history of the each taxa. Branches corresponding to partitions reproduced in less than 50% of the bootstrap replicates were collapsed. Each tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Evolutionary analyses were conducted in MEGA5Iterative Threading ASSEmbly Refinement) Suite for protein structure and function prediction444548The amino acid sequences for major virus proteins of the Dunlop (1a) reference virus subtype was downloaded from NCBI reference genome. The assembled contigs which resulted in analysis of complete genomes of B9/RU13 (IV) and B12/A-62H (1a), were used for the open reading frame (ORF) prediction using the NCBI ORF Finder suite. The predicted ORFs for VP1, VP2, and Large T antigen were then confirmed using the BLASTp suite, and sequences were retrieved for further analysis. The protein structure and residue binding sites for Dunlop (1a) and other BKV sequences were predicted using the I-TASSER .


The Formyl-peptide receptor-2 (FPR2) is a seven transmembrane G protein-coupled receptor, which plays an important role in sensing of bacteria and modulation of immune responses. FPR2 is also used by viruses for their own profit. Annexin A1, one of the multiple ligands of FPR2, is incorporated in the budding virus membrane of influenza A viruses (IAV). Thereby, once IAV infect a host cell, FPR2 is activated. FPR2-signaling leads to an increase in viral replication, a dysregulation of the host immune response and a severe disease. Conversely, experiments using FPR2 antagonists in a preclinical model of IAV infections in mice showed that blocking FPR2 protects animals from lethal infections. Thus, FPR2 represents a very attractive host target against influenza. In this review we will give an overview on the pathogenesis of influenza with a focus on the role of FPR2 and we will discuss the advantages of using FPR2 antagonists to treat the flu. Influenza virus infection is one of the most important infectious diseases affecting the respiratory tract , which in majority are nucleic acids that are not typically present in the host cells  and those of M2 (amantadine and rimantadine) Ison, . RegardiRegarding the viral NA, it plays a critical role for virus transmission from cell to cell. The first step of the influenza virus replication cycle is the binding of the virion to the host cell. This occurs through the interaction of the viral HA protein with sialic acids on the membrane of the host cell. The NA is a glycoside hydrolase enzyme that removes influenza virus receptor-binding sites and enables the newly synthesized virions to detach from the infected cells at the end of the viral life cycle. It also prevents virus self-aggregation. Thus, virions grown in the presence of NA inhibitors form aggregates near the cell surface, preventing virus spread  are a family of seven transmembrane domains receptors coupled to G protein. While three different FPR were described in humans (FPR1-3), at least eight FPR exist in mice (mFPR), designated FPR1 (FPR1), FPR-rs1 (FPR3 or LXA4 receptor), FPR-rs2 (FPR2), FPR-rs3, FPR-rs4, FPR-rs5, FPR-rs6, and FPR-rs7  is expressed by many cells, including epithelial and endothelial cells, fibroblasts, most if not all immune cells as well as neuronal cells. It binds several kinds of ligands , resolvin D1 or the glucocorticoid-modulated protein Annexin A1, allowing a resolution of inflammation and return to homeostasis. The concept that inflammation resolves through active processes is now commonly accepted , the day of infection or 1/2 days post-infection (treatment). Typically, using a lethal dose of A/PR/8/34 (H1N1) virus infection, 100% of mice reached the experimental endpoints while only 20–40% attained the endpoint after BOC2 or WRW4 treatment , PBP10  and BOC-2 (tert-butoxycarbonyle-FLFLF-OH). WRW4 is a six amino acid peptide which specifically impairs FPR2-signaling. It blocks the binding of agonists to FPR2 and thereby its downstream signaling pathway (Bae et al., Host factors represent useful targets for therapy to overcome the challenge of virus resistance. Some interesting molecules have been identified and this approach appears particularly relevant to treat influenza. The first class of novel promising antivirals are related to their capacity to block cellular functions supporting the virus life cycle. Many targets with antiviral properties were identified, including inhibitors of cytoskeleton, autophagy, proteasome, nuclear export or regulators of transcription (de Chassey et al., Another class of molecules aims at the protection of the tissues from damage induced by excessive inflammation. This novel approach concerns mainly all molecules with anti-inflammatory properties. These molecules could benefit patients with severe influenza at later stages post-infection but would not act on viral replication. In this regard, molecules such as statins (Kwong et al., kB or ERK (Pinto et al., In contrast, novel opportunities are currently emerging with the novel class of drugs that both inhibit virus replication and temper inflammation. For example, the antagonists of Protease-activated receptor-1 (Khoufache et al., Preclinical studies have proven that FPR2 antagonists efficiently protect mice against IAV infections, by inhibiting viral replication and deleterious inflammation of the lungs. FPR2 is a host receptor and thus targeting such protein is of particular interest in order to limit the emergence of IAV resistance. In addition, FPR2 antagonists will most likely generate a long lasting protection since it tempers inflammation which is responsible for tissue injury at later stages of the infection. Used together with oseltamivir, FPR2 antagonists might also have a much stronger effect in blocking IAV replication. Altogether by inhibiting viral replication and protecting the lungs from destruction, FPR2 antagonists appear as an appealing strategy to treat or prevent influenza in the future.All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.BR received funds from the patent valorisation SATT-Sud Est organization. The other 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.


Cost reduction measures in medicine are gaining greater importance nowadays, especially in high-volume procedures such as laparoscopic appendectomy (LAE). Currently there are two common methods of dissecting the appendix from the caecal pole: linear stapler and endoloops. The endoloop is the cheaper device but can only be used in uncomplicated cases of appendicitis. Therefore both methods are used in LAE depending on intraoperative findings. The goal of this study was to retrospectively evaluate possible cost reduction due to increased use of endoloop in LAE in our general surgery department of a tertiary referral university hospital.We previously used the stapler for appendix dissection in LAE as our local protocol but introduced the endoloop as standard method in 2015 to reduce intraoperative costs. We conducted a retrospective analysis of patients who underwent LAE between June 2014 and October 2015 in our department. Our purpose is to show the effects on cost reduction during the introductory period adjusting for a potential bias due to the individual learning curve of every surgeon. We estimated costs for LAE by taking into account average device costs and duration of operation (DO) as well as patient outcome.p < 0.001). The use of endoloops increased from 10% to 55% (p < 0.001). Patients’ characteristics and outcomes did not differ significantly. A median saving of 5.9€ per operation was calculated in phase II compared to phase I (p = 0.80).A total of 177 patients underwent LAE, 73 in 2014 (phase I) and 104 in 2015 (phase II). The median DO was 61 (± 24 SD) min during the entire period, and increased by 14 min from phase I to II (from 51 (±23 SD) min to 65 (±24 SD) min respectively, Introducing the endoloop as standard device for LAE leads to a marginal reduction in intraoperative costs without increasing negative outcomes. In our model the cost-reduction achieved by cheaper devices was overcome by increased costs for DO during the initial phase of use of endoloops. A longer follow up might show a more pronounced cost reduction. Acute appendicitis has an incidence of 100/100,000 per year in Western Europe, of which about 20% have already perforated when admitted to hospital  with aboSeparating the appendix from the caecum can be achieved using different devices: The most common one is the linear stapler because it can also be used in more advanced stages of appendicitis, e.g. with inflammatory infiltration of the caecal pole. Alternatively, ligation of the appendix can be performed using an endoloop with absorbable sutures. Both methods have been proven efficient and safe –7 althouThe application of an endoloop requires a more thorough preparation of the appendix and its mesoappendix and utilising an endoloop is more difficult than a stapler , 6. FurtThe German DRG (Diagnosis Related Groups) system reimburses LAE with a standardised amount irrespective of the method used for dissecting and resecting the appendix. The reimbursement is calculated by the average costs and average length of stay (LOS) in German index hospitals. With this system in place a patient has to be discharged at a lower to mean LOS in order to be profitable for the hospital. In LAE the target LOS is between the 2nd and 4th post-operative day. Besides an early discharge date, faster surgery using cheaper instruments in theatres is another way to increase profit. One stapler including one charge of staples costs about 280€, whereas using endoloops results in costs of about 45€ per usage. This results in a cost difference of about 235€. By using other disposable devices like the Harmonic Scalpel or LigaSure costs may increase but DO is reduced .The timing of an individual surgery is crucial as it should weigh the benefit of early surgery against possible drawbacks of night time surgery and its subsequent influence on costs –15.The purpose of this retrospective study was to analyse the extent to which it is possible to use the endoloop in LAE as a standard device in regards to the different stages of appendicitis. We furthermore wanted to analyse if the introduction of the endoloop for LAE as standard device is appropriate to reduce intraoperative costs. As main budget factors we considered material costs and costs for DO in order to calculate an approximate overall cost for comparison of endoloop versus stapler.At our university tertiary referral teaching hospital, we introduced the endoloop as standard technique by January 2015 with the intention to reduce intraoperative costs. A retrospective analysis of all patients receiving LAE between June 2014 and October 2015 was carried out with June to December 2014 being phase I and January to October 2015 representing phase II. To analyse the immediate effects of introducing the endoloop as standard device we included a limited number of patients after its introduction in phase I and matched the group size with an approximately equal number of patients from the period prior to the introduction of the endoloop. We chose this approach accepting a degree of bias due to the individual learning curve of every surgeon to evaluate possible immediate effects of the new standard technique.Data on age, sex, method of resection (stapler or endoloop), histological findings, DO, post-operative LOS, as well as post-operative complications before and after introduction of endoloop were collected. Additionally, we determined surgeons’ experience by using our respective group of duty as a surrogate, i.e. young (1st to 4th year)/old residents (~5th to 6th year), specialists and consultants. Furthermore, the time of day the operation took place  was recorded to evaluate its influence on DO.We use a three trocar approach with multi-use trocars and standardised sutures. In phase I the stapler was the standard approach; in Phase II the endoloop was the standard device but if an advanced appendicitis with inflammation of the proximal appendix, the caecal pole or perforation was encountered, resection was performed with the stapler. For skeletonization and preparation of the appendix monopolar cautery is used. It is up to the operating surgeon’s discretion which device to use according to the criteria mentioned above. Once intraoperative decision on method of resection is made the respective device is opened. From the operating notes it is not possible to conclude if redundant endoloops or stapler firings were used and we therefore performed calculations assuming the reported number was opened and then used.We use the 35 mm ETS Articulating Linear Cutter  which costs about 280€ with one set of staples, an extra staple set costs about 160€. The endoloop is an absorbable PDS II, suture strength 0 endoloop (Ethicon Endo Surgery) of which 3 loops are required , resulting in costs of about 45€ per case. One minute of operating room (OR) time cost about 12€ in our institution in 2015. This is a mean calculated for day and night time, including costs for anesthesia and room cleaning and standard materials such as sutures or drapings. From those figures the average cost of a procedure was calculated by adding costs of OR time and material costs.We did not intend to perform a cost-consequence analysis or a cost-effectiveness analysis as those models do not seem appropriate to us for this certain setting.Follow-up conducted was limited to the post-operative hospital stay as further routine follow-ups are performed by a patient’s local general practitioner. We therefore only included in-hospital complications or complications registered by readmission to our department.2 test were used. As costs showed a standard distrubution we used the unpaired t-test. A multivariate analysis was performed to determine to which extent DO is influenced by method of dissection, surgeon’s experience, time ofn day and patient age. P-values were set at 0.05 and graphs were created using SPSS 20.Patient data was collected from electronic patient charts, assembled in Excel 2010, version 14.5.1  and analysed in SPSS 20 . For all continuous variables, the median and standard deviation was calculated and therefore Mann-Whitney-U test (MWU test), Kruskal-Wallis test or XOne hundred seventy seven patients underwent LAE between June 2014 and October 2015. Demographics and histological findings are listed in Table P < 0.001); the use of endoloops increased significantly in phase II .A significant difference could be found for DO: 51 min (±23 min SD) in phase I vs. 65 min (±24 min SD) in phase II (P = 0.29), sex (P = 0.28), histologic findings (Table P = 0.27) and post-operative LOS (P = 0.38). In phase I there was a trend towards shorter DO with increasing experience which was not found in phase II. None of those were statistically significant with p = 0.82 and p = 0.23 for phase I and II respectively. No significant difference was observed for the time of day at which the operation was performed (P = 0.61). The X2-test could not demonstrate a significant difference between which group of surgeons preferred a certain method, endoloop or stapler (P = 0.13).In both phases, no significant differences were noted with regard to patients’ age , ranging from superficial wound site infection in one patient on immunosuppressants after liver transplantation to persistent intraabominal abscess due to stumpf insufficiency after stapler appendectomy that required reintervention. We could not identify a device specific increase in complications in phase II. In both groups about one third of histologically or macroscopically perforated appendicitides caused intraabdominal abscess formation. In phase II also 3 histologically intact but highly active ulcero-phlegmonous appendicitides caused perityphlitic abscesses. Perityphlitic abscesses were treated with postoperative abdominal lavage via intra-operatively placed drainage catheters and intravenous antibiotics. One patient died due to severe sepsis at presentation to our emergency department which did not improve despite resecting the necrotic appendix. Another patient suffered from severe sepsis post-operatively due to a perforated necrotic appendicitis but consequently fully recovered.Surgical complications were distributed equally for both phases  per patient when endoloops are used instead of the linear stapler presuming that in every case the endoloop could be used. However, the DO is another crucial factor influencing the total surgical costs of LAE. Considering both those factors we calculate average costs per procedure by adding costs per device and per time in the OR. The average costs per phase and device are summarised in Table p = 0.80) while material costs decreased by 105.4€ (p < 0.001). Table Since there was no exclusive use of either device in phase I and II as surgical indications vary as described above we calculated the average total cost difference for all procedures performed in the two phases which resulted in 5,90€  costs for DO and ii) average material costs for resection devices. Therefore minimising both of them will result in the lowest intraoperative costs possible.We found an increase in DO after introduction of the endoloop (from 51 ± 23 min SD in phase I to 65 ± 24 min SD in phase II). This increase is also present when analysing the surgeons’ sub-groups although the correlation is not as pronounced in phase II compared to phase I. DO in laparoscopic appendectomy varies considerably in literature independant of the device used. No reports exist so far how operative experience influences DO. DOs between 50 and 62 min have been described for stapling ; for theThe increase of DO in phase II may be a temporary phenomenon: the introduction of endoloop represented a new technique even for many of our specialists. A learning curve was therefore expected as in our personal experience performing roughly 3 cases with endoloops was required to learn proper handling and placement of the loop. The endoloop procedures performed in phase I were exclusively performed by one specialist who had previously worked with this device in another hospital. Therefore these DOs are not representative or comparable to those in phase II. The other surgeons had never used endoloops before. Further follow-up in this regard is mandatory and may elucidate the bias by learning the new technique.In our study DO does not vary over the course of the day. There is a tendency towards an increase in DO towards later hours Fig.  which isThe second variable in our study is the intended reduction of intraoperative costs for devices. The more frequent use of endoloops, rising from 10% in phase 1 up to 55% in phase II meant that the remaining difficult cases were reserved for the use of the stapler device. This is reflected by the longer DO and LOS in the stapler subgroup of phase II Table . The intA faster recovery and earlier discharge have been demonstrated in LAE compared to open surgery . No diffBy using the aforementioned approach we calculated intraoperative savings of 5.9€ per LAE in 2015 compared to 2014 Table . When caConsidering all patient characteristics, intraoperative and histological findings as well as surgeon’s experience did not differ significantly the only variable that explains the pronounced increase in DO is the difference in device used. The increase in DO almost negates the financial benefits of using the cheaper device. As complications and post-operative LOS did not differ between both phases we suggest that consequently intraoperative costs are the biggest variable contributing to the total costs in a case of acute appendicitis. The cost calculations presented herein are only rough estimations and only provide a trend as costs are not routinely monitored per patient in the hospital (e.g. Day Mix Index) and costs of devices and time in the OR may vary significantly between hospitals and countries. Additionally our data represents the introductory period of the new technique thus the results might be influenced by the surgeons’ inexperience that leads to a relatively greater increase in duration of operation. To eliminate this effect a much longer study period is needed. As our intention was to show direct effects on costs we only chose the limited time frame.All these limitations of our study taken into consideration: We are the first to report the direct effects on intraoperative costs upon introducing the endoloop as standard method for resecting the appendix in LAE. Our results may be biased by every surgeon’s inexperience with the endoloop in the beginning and in relation to surgeons’ individual learning curves. We propose a training duration of about three cases to be able to place the endoloop without any technical difficulties. A longer prospective follow-up period is required to fully evaluate the influence of the learning curve and the steady state once the technique has been mastered to possibly demonstrate greater advantages of the endoloop. Additionaly limiting the patient inclusion criteria to early to mild appendicitis with randomised use of either endoloop or stapler could elucidate financial benefits but would then not reflect clinical reality.Although the endoloop is a practical and cheap device for resecting the appendix in laparoscopic appendectomy, the “true” economic benefit of the endoloop as standard method for LAE is not as high as expected a priori when regarding costs of each device. In our series, costs by prolonged DO negated some of the expected savings by endoloop for various possible reasons, e.g. surgeon’s learning curves, surgeons at an early stage of training. Growing experience with the endoloop might reveal more pronounced cost-reduction possibilities.