Well‐preserved molecular cargo in circulating extracellular vesicles (EVs) offers an ideal material for detecting oncogenic gene alterations in cancer patients, providing a noninvasive diagnostic solution for detection of disease status and monitoring treatment response. Therefore, technologies that conveniently isolate EVs with sufficient efficiency are desperately needed. Here, a lipid labeling and click chemistry‐based EV capture platform (“Click Beads”), which is ideal for EV message ribonucleic acid (mRNA) assays due to its efficient, convenient, and rapid purification of EVs, enabling downstream molecular quantification using reverse transcription digital polymerase chain reaction (RT‐dPCR) is described and demonstrated. Ewing sarcoma protein (EWS) gene rearrangements and kirsten rat sarcoma viral oncogene homolog (KRAS) gene mutation status are detected and quantified using EVs isolated by Click Beads and matched with those identified in biopsy specimens from Ewing sarcoma or pancreatic cancer patients. Moreover, the quantification of gene alterations can be used for monitoring treatment responses and disease progression. This work demonstrates an efficient and rapid extracellular vesicle (EV) capture platform (“Click Beads”), which enables downstream quantification of gene alterations in both Ewing sarcoma and pancreatic cancer using reverse transcription digital polymerase chain reaction (RT‐dPCR). The streamlined workflow that combines Click Bead‐based EV capture and RT‐dPCR exhibits potential clinical utility in disease detection and treatment response monitoring. EVs are produced from all cell types and can be found in body fluids and blood circulation, making these nanoparticles one of the three liquid biopsy applications. Ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and protein are the biomolecular cargoes encapsulated in EVs, which mirror those in their tissues or organs of origin. The detection and characterization of EVs and their cargoes could introduce a rich source of circulating biomarkers for disease diagnosis and treatment monitoring in a noninvasive manner. Over the past two decades, a large collection of technologies has been developed to enrich, detect, and analyze EVs.Extracellular vesicles (EVs)1 are  filtration, precipitation, and size‐based microfluidic enrichment, were developed to isolate EVs in blood based on their physical properties . However, the ultracentrifugation is not convenient and requires large instrument, and the reported performance of different technologies for EV isolation varies widely. Therefore, technologies that conveniently isolate EVs with sufficient efficiency are desperately needed. Novel techniques have been developed for more efficient and convenient EV enrichment methods with the implementation of downstream molecular characterization. Zheng et al. developed a powerful EV enrichment technology, which combines lipid labeling and magnetic beads to effectively tag and recover EVs from plasma samples. This allows for downstream analysis of mutations in epidermal growth factor receptor (EGFR) and kirsten rat sarcoma viral oncogene homolog (KRAS) genes in nonsmall‐cell lung‐cancer patients. The bilayer membranes are the most prominent characteristics of EVs, and labeling EV membranes by the insertion of lipophilic membrane dyes  is the most common way to characterize EVs. The use of lipid labeling allows for EV capture independent of surface antigens, providing the flexibility to capture and enrich all EVs. Building upon this approach, we aim to maximize the utility of lipid‐based labeling for EV capture technologies.Conventional technologies, such as ultracentrifugation,7 filtFiguresn‐glycero‐3‐phosphoethanolamine‐polyethylene glycol (DSPE‐PEG1000‐TCO) into the EV membranes. The DSPE‐PEG1000‐TCO conjugate was prepared by incubating TCO‐PEG‐NHS ester with DSPE‐PEG1000‐NH2, as shown in Scheme  In this study, Click Beads with DSPE‐PEG1000‐TCO enables effective capture of EVs derived from cancer cells of multiple origins, such as epithelial  and mesenchymal  origin, since lipid labeling bypasses the need for surface antigens. We adopted this streamlined workflow to quantitatively detect gene alterations  gene rearrangements in Ewing sarcoma and KRAS mutations in pancreatic cancer patients. We demonstrated that the Click Beads‐based universal capture of total EVs, combined with cancer‐specific gene alteration detection and quantification, allows for noninvasive cancer diagnosis and treatment monitoring.We introduce a rapid EV enrichment solution that combines the lipid‐based labeling and click chemistry‐mediated EV capture onto microbeads , thus allowing for isolation of EVs via simple centrifugation. In a streamlined workflow Figure1, EVs 22.1Figure2‐modified microbeads. Subsequently, the NH2‐modified microbeads are treated with tetrazine‐polyethylene glycol‐N‐hydroxysuccinimide (Tz‐PEG‐NHS) ester to yield Click Beads. Surface composition analysis using X‐ray photoelectron spectroscopy (XPS) for Click Beads preparation was shown in Figure To enable a rapid workflow for capture and characterization of EVs, we prepared Tz‐grafted microbeads, i.e., Click Beads, which are capable of immobilizing TCO‐labeled EV via a bioorthogonal click chemistry reaction. EVs from A673 cells (an Ewing Sarcoma cell line) were harvested via ultracentrifugation and identified by transmission electron microscopy (TEM), dynamic light scattering (DLS), and nanoparticle tracking analysis (NTA). The EVs imaged with TEM exhibited cup morphologies, as shown in Figure After immobilizing A673 cell‐derived EVs onto Click Beads using the workflow described in Figure 2.2Figure10 particles mL−1  were tested. 50 nmol was found to be the optimal concentration  of Click Beads. Once the amount of Click Beads exceeded 107 per study, there was no significant difference in the observed capture yields . We then checked the dynamic range of Click Beads using 1.0 mL artificial plasma samples spiked with different concentrations of EVs containing 0–1600 copies of EWS/FLI‐1 mRNA and confirmed the consistency of EV capture yields   of plasma samples, we demonstrated that EV capture performance Figure  was not ) Figure . Togethe1000‐TCO for capturing EVs from different types of tumors, a study was conducted to compare capture yields of this method with those of four other methods: Click Beads with anti‐CD63‐TCO, ExoQuick (System Biosciences), magnetic beads (Dynabeads) with biotin‐conjugated DSPE, and ultracentrifugation. In this study, four types of artificial plasma samples (1.0 mL per study), two spiked with EVs from Ewing Sarcoma cell lines , and the other two were spiked with EVs from pancreatic cancer cell lines , were prepared and used. For ES‐5838 EVs (with EWS/ERG rearrangement), CFPAC‐1 EVs (with KRAS G12V mutation), and AsPC‐1 EVs (with KRAS G12D mutation), the respective RT‐dPCR assays were developed to quantify the spiked and captured EVs. We then calculated the respective EV capture yields by adopting formulas (see the Supporting Information) similar to Equation (1000‐TCO exhibited superior capture yields (ranging from 73.1% ± 6.0% to 80.6% ± 3.6%) for all four cell line‐derived EVs in comparison to capture yields of Click Beads with anti‐CD63‐TCO (ranging from 14.5% ± 1.4% to 37.2% ± 0.1%). The latter technique exhibited consistently low capture performance due to the dependence of CD63 on EV surfaces. Meanwhile, the capture yields of the three control studies, including ExoQuick ULTRA EV Isolation Kit (ranging from 29.1% ± 2.7% to 51.2% ± 6.7%), magnetic beads using biotin‐PEG‐DSPE conjugates (ranging from 41.8% ± 3.9% to 57.5% ± 0.9%), and ultracentrifugation (ranging from 12.8% ± 6.1% to 18.2% ± 9.7%), were all lower than those of Click Beads with DSPE‐PEG1000‐TCO. Overall, these results further verified the reproducibility and general applicability of Click Beads with DSPE‐PEG1000‐TCO for capturing EVs, providing a framework for future studies using clinical samples.To confirm the reproducibility and general applicability of Click Beads with DSPE‐PEG2.41000‐TCO protocol, we then explored its clinical utility in Ewing sarcoma with an modified workflow to quantify the two most common subtypes of EWS gene rearrangements . No mutated KRAS was detected in the plasma samples of healthy donors. We monitored the dynamic changes in one pancreatic cancer patients (PanC03) during treatment by aligning the total copies of KRAS transcripts (obtained by following the modified workflow) and serum CA19‐9 levels (U mL−1) with CT images taken before and post‐treatment. Serial plasma samples were collected from patient PanC03 19 days prior to initiation of treatment and 84 and 210 days post‐treatment. The corresponding serum CA19‐9 levels and copies of KRAS transcripts for each timepoint were plotted in Figure We tested the clinical utility of the modified workflow Figure5A in pa31000‐TCO and click chemistry‐mediated EV capture onto Click Beads has been demonstrated to enable downstream molecular analysis of EVs. After conducting multifaceted optimization of the experimental conditions, we established a streamlined workflow which integrates Click Bead‐based EV capture and RT‐dPCR quantification of EV‐derived mRNA. This workflow was adopted to quantitatively detect gene alterations, i.e., EWS rearrangements in Ewing sarcoma and total KRAS transcripts including both mutated KRAS and wild‐type KRAS in pancreatic cancer, allowing for noninvasive cancer diagnosis and treatment monitoring.In this study, a rapid EV enrichment technology that combines the lipid‐based EV labeling using DSPE‐PEG the lipid‐based EV labeling by DSPE‐PEG1000‐TCO is independent of EV surface antigens. The insertion of the lipid motif of DSPE‐PEG1000‐TCO effectively and specifically grafts TCO onto EVs. In the presence of Click Beads, the TCO‐labeled EVs are instantaneously immobilized onto Click Beads via a bioorthogonal and irreversible click chemistry reaction between TCO and Tz motifs. Through rapid and simple centrifugation, the EVs captured on the Click Beads are collected. Overall, Click Beads with DSPE‐PEG1000‐TCO uniquely leverages the convenience of lipid‐based EV labeling and specificity and efficiency of click chemistry, introducing an effective EV capture method that preserves biomolecular cargoes  within the EVs for further analysis.In contrast to immunoaffinity‐based EV labeling which is constrained by the limited numbers of EV‐specific surface antigens  on the EV membranes,19 the This technological development has the potential to advance the standard of care of this disease, as bone tissue biopsies utilized in the routine pathological diagnosis of this disease undergo decalcification when preparing FFPE slides, which severely damages the quality of the mRNA available for downstream EWS genotyping, rendering it infeasible for mRNA detection in decalcified tissues. This clinical challenge is highlighted by the fact that out of 28 Ewing sarcoma patients, only 12 had satisfactory FFPE tissues from metastatic non‐bone tumor deposits that were able to undergo EWS rearrangement confirmation using RT‐dPCR. After the initial diagnosis, dynamic monitoring of clinical treatment response is needed. The Click Bead‐based EV capture method combined with RT‐dPCR provides a noninvasive diagnostic solution, offering a treatment‐response monitoring for Ewing sarcoma patients.The EV‐derived mRNA obtained from Click Bead‐based EV capture is of high quality and can undergo downstream RT‐dPCR assay. One application is the detection and quantification of EWS rearrangements in patients with Ewing sarcoma, an aggressive bone and soft‐tissue tumor that occurs primarily in the adolescent and young adult population.21 Thi were detected in majority of the pancreatic cancer patients and correlated with KRAS mutation status of the matched tissues. Three patients showed inconsistent KRAS status in tissue and paired plasma samples, and this probably could be attributed to the heterogeneity of the single biopsy tissue or the copy number changes of mutated KRAS over the period between tissue biopsy and blood draw. Wild‐type KRAS were detected for all patients, and the higher copy numbers of total KRAS transcripts were used to differentiate pancreatic cancer from healthy donor and monitor the disease progression of pancreatic cancer.Furthermore, we demonstrated the general applicability of Click‐Bead based EV capture method by expanding its clinical utility to pancreatic cancer. Here, three commonly‐seen KRAS mutations 17b we Recently, it has been reported that quantification of circulating EVs can be used for monitoring therapy efficiency in cancer. EVs can be a potential noninvasive diagnostic solution for detecting cancer of either epithelial origin  or mesenchymal origin .Overall, EVs captured by our Click Beads can be a potential noninvasive diagnostic tool for informing upfront treatment response or therapeutic effectiveness of salvage therapies, and relapse/recurrence monitoring for those cancer patients in remission. Although current and emerging techniques of radiologic imaging  have been used to detect tumor recurrence and evaluate treatment response.24 Rec Click Beads can be adopted in most of the research laboratories without specialized device setting to handle a broader range of volumes of plasma samples. We also note that our current study does have some limitations, for example limited cohorts for the both tissue and plasma samples. Testing such a streamlined workflow using study cohorts with larger sample sizes will be implemented in our future plan.In conclusion, coupling lipid labeling and click chemistry enables effective isolation of EVs for noninvasive detection, characterization, and quantification of oncogenic gene alterations in both Ewing sarcoma and pancreatic cancer. Our streamlined workflow that combines Click Bead‐based EV capture and RT‐dPCR‐based quantification of oncogenic gene alterations showed potential clinical applications in detecting disease progression and monitoring treatment responses. Compared to our previously published EV purification method by EV Click Chips,15 Cli4Ewing sarcoma cell lines of A673, ES5838 or pancreatic cancer cell lines of CFPAC‐1, AsPC‐1 was cultured in 18 dishes (Thermo Scientific Nunc EasYDish Dishes) under usual conditions until 70–80% confluency. Next, cells were cultured with exosome‐production medium (13 mL per dish) for 24 h. A total of 228 mL conditional medium was collected and centrifugated at 300 g, 4 °C for 10 min followed by another centrifugation step at 2800 g, 4 °C for 10 min to discard cell debris. The medium was carefully transferred to Ultra‐Clear Tubes  and then ultracentrifuged at 100 000 g, 4 °C for 90 min. The captured EVs were suspended in 200 µL PBS as original EV samples. For the artificial plasma samples, each 10 µL aliquot of EV pellets was spiked into 90 µL healthy donors’ plasma.8 beads, 2.5 µm in diameter and 2.0 g cm−3 of density) were first washed through an acid incubation  for the regeneration of hydroxyl groups, and followed by immediate silanization in an ethanol solution (600 µL) containing 4% (3‐aminopropyl)triethoxysilane  for 45 min at room temperature. The amino‐functionalized silica microbeads were washed with ethanol three times to remove the unbound silane and then reacted with Tetrazine‐PEG‐NHS ester  in DMSO/PBS  for 1.0 hr.The Click Beads  in PBS (100 µL) was used to treat Click Beads  for 30 min. Prior to use, the Click Beads were rinsed with deionized water five times to remove any unbound Cy5‐labeled TCO. Fluorescence images were collected with a Nikon 90i fluorescence microscope .Cy5‐labeled TCO reagent (5 × 101000‐TCO conjugate was prepared by incubating TCO‐PEG‐NHS ester (5.0 × 10−3 m) with DSPE‐PEG1000‐NH2 (5.0 × 10−3 m) in a DMSO/PBS solution at room temperature for 30 min. The DSPE‐PEG1000‐TCO solution was stored at −20 °C until use.A solution of DSPE‐PEG1000‐TCO were mixed with artificial plasma samples for 5–20 min at room temperature, and TCO‐grafted EVs plasma samples were obtained.10 µL of original EVs were added to 90 µL  of plasma  to prepare artificial plasma samples, and the amount of EVs spiked into each sample was standardized by RT‐dPCR. Then 10, 50, 75, 150 nmol of DSPE‐PEGThe plasma samples containing TCO‐grafted EVs were incubated with Click Beads for 5–30 min followed by a centrifugation at 5000 g for 2 min. In different EV isolation methods testing, the respective optimal capture conditions were applied for Click Beads (DSPE‐TCO and anti‐CD63‐TCO (50 ng/100 µL) and DSPE‐biotin magnetic beads. Additionally, 100 µL of artificial plasma sample with the same amount of original EVs was added to 38 mL PBS and ultracentrifuged once at 100 000 g and 4 °C for 90 min. 100 µL of artificial plasma samples with the same amount of original EVs were used as the input for ExoQuick tests, the EVs were purified according to the manufacturer's protocol.For SEM, EVs captured onto Click Beads were fixed in 4% paraformaldehyde  for 1 h. The samples were then sequentially immersed in 30%, 50%, 75%, 85%, 95%, and 100% (twice) ethanol solutions for 10 min per solution, followed by sputter‐coating with gold at room temperature. The morphology of the EVs was examined using a ZEISS Supra 40VP scanning electron microscope.For TEM, ultracentrifuged EV samples were fixed in 4% PFA (in PBS) for 30 min. Then the samples were dropped on a 400‐mesh carbon‐coated copper grid and incubated for 10 min at room temperature. Excess samples were blotted with filter paper and washed 5 times by water. Grids were dried for TEM imaging by a T20 iCorr (FEI) high‐resolution cryo‐TEM at an accelerating voltage of 80 kV. For immunogold staining, fixed EVs in PBS or captured on Click Beads were incubated with anti‐CD63  for 30 min. Then, these samples were incubated with antimouse nanogold  for 1 h. The gold‐labeled samples were dropped onto carbon coated copper grids and incubated for 10 min before being wiped off from the grids. After being rinsed 5 times using water, grids were then dried for TEM imaging. The ultra‐centrifuged EV samples (10 µL) were diluted into 1 mL in PBS for DLS and NTA tests.The captured EVs on Click Beads were lysed using a QIAzol lysis reagent. A miRNeasy mini kit  was used to extract and purify RNA from the isolated EVs. The RNA concentration in the EVs was measured using a Qubit 3.0 Fluorometer  in combination with the Qubit RNA HS Assay  according to the manufacturer's protocol.The collected RNA (9.4 µL) was reversely transcribed to complementary DNA  using a Thermo Scientific Maxima H Minus Reverse Transcriptase Kit . Complementary DNA was synthesized under the condition of 55 °C for 30 min and 85 °C for 5 min, and then preamplification was performed using 4.0 µL of cDNA as input to obtain 10 µL preamplified product for each gene according to the manufacturer's instructions of Prelude PreAmp Master Mix Kit (Takara Bio).For dPCR, the reaction mixture (40 µL) including 4 µL of preamplified product was loaded into each well of a nanoplate . The nanoplate was transferred into the QIAcuity instrument  for the following PCR process. A programmed Thermal Cycler was set at 95 °C for 2 min followed by 40 cycles of 95 °C for 15 s and 60 °C for 30 s. The readouts of positive and negative partitions were counted automatically by the instrument and analyzed via QIAcuity software.1000‐TCO at room temperature for 10 min before incubation with Click Beads for EV capture.Peripheral venous blood samples were collected from patients or healthy donors with written informed consent according to the institutional review board protocols (IRB #14‐000 197) at UCLA. Each 10 mL blood sample was collected in a BD Vacutainer plastic tube  with EDTA. Samples were processed according to the manufacturer's protocol within 4.0 h of collection. The final plasma samples were collected after centrifugation at 10 000 g for 10 min and aliquoted and stored in ‐80 °C refrigerators. Each plasma samples were centrifuged at 10 000 g for 10 min after thawing at 37 °C. 1.0 mL plasma samples were then incubated with DSPE‐PEGL‐lysine coated glass slides. EWS rearrangements analysis of tissues from Ewing sarcoma patients and KRAS mutation analysis of tissues from pancreatic cancer patients were performed. Total RNA was extracted from FFPE tissue sections using Qiagen RNeasy FFPE kit , followed by RT‐dPCR.Serial 4 µm thick tissue sections from FFPE blocks were cut and mounted on poly‐Following the management plan provide by UCLA Conflict of Interest Review Committee, Dr. Hsian‐Rong Tseng would like to disclose that (1) he has a financial interest in CytoLumina Technologies Corp. and Pulsar Therapeutics Corp., and (2) the UC Regents have licensed intellectual properties invented by Dr. Tseng to CytoLumina and Pulsar. Other authors declare no conflict of interest.N.S., Y.Z., and H.R.T. designed and performed most of the research and data analysis. N.S. performed the preparation and optimization of Click Beads. N.S. and P.Y. performed DLS, Zeta potential, SEM and TEM tests. J.S., H.Y., J.S. performed XPS analysis. B.T. and Z.P. performed the collection of blood samples and clinical information with assistance from S.J., J.T., N.F., and S.L. N.S., Z.P., J.W., C.Z., and R.Z. performed blood sample tests. N.S. and C.Z. performed FFPE sample testing. N.S. and Y.Z. made schematic diagrams and figures with assistance from T.Z. W. X., T.Z., J.W., N.S., Y.Z., and H.R.T. wrote and revised the manuscript with input from A.K., H.Z., V.A., R.P., J.T., J.T., S.J., and N.F. Y.Z. and H.R.T. oversaw project execution.Supporting InformationClick here for additional data file.


In Yangquan County, the sandfly-transmitted virus (Wuxiang virus) was first isolated from sandflies in 2018. However, relationships between the abundance and seasonal fluctuations of local sandflies and sandfly-transmitted viruses are unknown. Herein, we report that sandfly specimens were collected in three villages in Yangquan County, from June to August, 2019. A total of 8363 sandflies were collected . Eighteen virus strains  were isolated in pools of Phlebotomus chinensis. The genome sequence of the newly isolated virus strain was highly similar to that of the Wuxiang virus (WUXV), isolated from sandflies in Yangquan County in 2018. Our results suggested that the sandfly-transmitted viruses, and the local sandfly population, are stable in Yangquan County, and that June is the peak period for the virus carried by sandflies in this area. The Phlebovirus genus belongs to the family Phenuiviridae and the order Bunyavirales . In MarcThe transmission vectors of phleboviruses include sandflies, mosquitoes, ticks, and other arthropods . PhlebovIn the summer of 2018, we isolated Wuxiang virus (WUXV) from sandfly specimens collected in Yangquan County, Shanxi Province, Central China. This was the first time the phlebovirus was isolated from sandfly specimens collected in rural areas of Yangquan County . Howeverhttps://guangzhou.11467.com/info/8049209.htm (accessed on 27 November 2022)). The specimen collection period was from 18:00 of one day to 06:00 of the next day. All collected sandfly specimens were placed in a low-temperature refrigerator for 30 min and then transferred to an ice bath, where the sandflies were classified according to insect morphology, collection time, and collection environment, among other criteria. All collected specimens were stored in liquid nitrogen until laboratory testing [Three villages in Yangquan county, Shanxi Province, China were selected for sampling  for the  testing ,11,12,133, in a 37 °C incubator with 5% CO2. C6/36 cells were cultured in 89% Roswell Park Memorial Institute Medium 1640 (Invitrogen), supplemented with 10% FBS and 1% penicillin and streptomycin (100 U/mL), in a 28 °C incubator [BHK-21 cells (Golden hamster kidney cells) and C6/36 cells  were cultured for viral infection analysis. BHK-21 cells were cultured in 90% Eagle’s minimal essential medium (laboratory preparation), supplemented with 7% fetal bovine serum , 1% penicillin and streptomycin 100 U/mL), 1% glutamine (30 g/L), and 1% NaHCO0 U/mL, 1ncubator ,12.3). Next, 1.5 mL grinding fluid was added to the samples, which were ground under ice bath conditions until the specimens were completely homogenized. The samples were centrifuged , and 100 µL of the ground supernatant was inoculated with an 80% confluent monolayer of BHK-21 cells or C6/36 cells on culture plates . The inoculated BHK-21 and C6/36 cells were grown continuously and monitored for cytopathic effects (CPEs) under a microscope every 12 h. The supernatants of cell cultures exhibiting CPEs were collected and stored at −80 °C for viral identification. The supernatants of first-generation cells without CPEs were blindly passed through both BHK-21 and C6/36 cells for three generations and those that did not cause CPEs were discarded [Pools of 50–100 sandflies were added to a glass grinder and washed twice with grinding fluid , according to the manufacturer’s instructions. The extracted RNA was immediately incubated in a 65 °C water bath for 10 min and then quickly transferred to an ice bath for 2 min. RNA (32 µL) was added to the first-strand reaction tube of the Ready-To-Go kit . The reaction tube was incubated at room temperature for 1 min, and then 1 µL random primer (pd(N)6)  was added, before further incubation at 37 °C for 1 h. The total volume of the resulting cDNA library was 33 µL, which was used immediately or stored at −40 °C for further use ,15.® GreenMasterMix , and 10 µmol/L each primer (forward and reverse). After PCR, 5 µL of the amplification product was examined by 1% agarose gel electrophoresis. The nucleotide sequences of the WUXV-positive amplification products were then determined by first generation Sanger sequencing [WUXV-specific primers  were analyzed by 1% agarose gel electrophoresis. The nucleotide sequences of the virus-positive amplification products were determined [Viral genes were amplified by first generation Sanger sequencing by PCR in a 25 µL reaction volume comprising cDNA template, 2× GoTaqtermined ,15. The The viral nucleotide sequences were aligned using the basic local alignment search tool . Seqman software  was used to assemble the nucleotide sequences and analyze read quality. Nucleotide multiple sequence alignment was performed using BioEdit software . MEGA6.0 software was used to conduct phylogenetic analyses using the neighbor-joining method with a bootstrap value of 1000, and identity analyses of nucleotide and amino acid sequences were performed using MegAlign software ,15. ExceThe DNA of WUXV-positive sandfly specimens was extracted and subjected to PCR amplification targeting the mitochondrial cytochrome c oxidase I gene  using thAssuming that each positive pool contained only one infected sandfly, the minimum infection rate was calculated as follows: minimum infection rate of 1000 sandflies = number of positive specimen pools (number of infected sandflies)/total treated sandflies × 1000 .We conducted four specimen collections at six fixed collection points in three villages in Yangquan County, Shanxi Province, China shown in  on 11 JuThe 8363 collected sandfly specimens were divided into 106 pools for grinding treatment. Of the 106 sandfly supernatant pools, 18 triggered CPEs in BHK-21 cells and were stably passaged . BHK-21 Phlebotomus chinensis. No sequence information for other sandfly species was found in 18 pools.To identify the sandfly species in which WUXV was detected, the 18 WUXV-positive sandfly pools were subjected to genetic testing. We found that 18 WUXV-positive sandfly species were We determined the nucleotide sequences of the coding regions of the L, M, and S segments of the isolated virus strains. The number of nucleotides (number of amino acids) of the coding regions of the L, M, NS, and N genes of the 18 virus isolates were 6273 nt (2090 aa), 4089 nt (1362 aa), 783 nt (260 aa), and 741 nt (246 aa), respectively. The L, M, and S segment lengths were the same between the WUXV strain isolated in the present study (SXYQ1944-2) and that isolated previously (SXWX1813-2) .Nucleotide sequence alignment of the M, NS and N segments of each of the 18 virus isolates revealed nucleotide and amino acid sequence identities of 96.5–99.8% (97.1–99.8%), 94.6–100% (97.3–99.99%) and 98.1–100% (98.8–99.6%) among the isolates, respectively . HomologThe genome sequences of the 18 virus strains isolated in this study were subjected to molecular phylogenetic analysis by comparing the L, M, and S genomic segments with those of 60 sandfly viruses recognized by ICTV. The 18 viruses isolated in this study clustered in the same phylogenetic branch as the WUXV strain SXWX1813-2 , providiIn this study, a group of WUXV virus strains was isolated from sandfly specimens collected in rural areas of Yangquan County in 2019.Phlebotomus chinensis is the dominant species of sandflies in Yangquan County [P. chinensis. P. chinensis sandflies reside in near-wild habitats, breed in the wild, and inhabit nearby village livestock houses, where they feed on the blood of cows, donkeys (mules), chickens, and domestic dogs [P. chinensis in Sichuan Province, Western China [P. chinensis in Yangquan County, Shanxi Province, China was examined, revealing that 88.9% and 66.7% of sandflies contained chicken and human blood, respectively [P. chinensis in Yangquan County. Given that multiple virus strains were isolated from sandflies in Yangquan County, a seroepidemiological investigation into sandfly-transmitted viruses in local human and animal populations is needed. This would enable evaluation of the infection prevalence and public health significance of sandfly-transmitted viruses.Yangquan County  is located in Shanxi Province in Central China on the west side of the central Taihang Mountains, with an average elevation of 700–1700 m and a warm temperate continental climate. Ecological environments that are arid, with low rainfall and high altitudes, are suitable for the growth and reproduction of sandflies, mosquitoes, and other blood-sucking insects ,18. Howen County ,20,21. Ttic dogs . Pigs arrn China . Recentlectively , suggestP. chinensis specimens in this study, was closely related to the Toros virus isolated from sandflies in Turkey in 2012 [We performed molecular phylogenetic analyses of the 60 Phlebovirus species recognized by ICTV in 2020 and the 18 virus strains isolated from sandflies in Yangquan County in this study. We found that the L, M, and S genomic segments each formed a common phylogenetic branch with a WUXV strain, previously isolated in China , and a v in 2012 .The PCR primers used to amplify the original WUXV genome were initially applied to amplify the genome of the virus strain isolated in this study. The nucleotide sequences of the three genomic segments of sandfly virus isolated here and the original WUXV isolated in the Wuxiang area exhibited differences, which might explain the poor amplification of the strains of newly isolated WUXV genomes using the primers designed for the original WUXV isolate . MoreoveLeishmania spp., a parasite that causes the severe sandfly-transmitted disease kala-azar, is endemic in Yangquan County [In this study, we combined field surveys of rural sandflies in Yangquan county with cell culture and molecular methods to isolate and identify sandfly viruses. The primary host species of the WUXV strain, and the prevalence of the virus in humans and animals, requires further study. The bloodsucking habits of sandflies also require further exploration. n County . The pot


Background: Next-generation sequencing (NGS) of tumor genomes has changed and improved cancer treatment over the past few decades. It can inform clinicians on the optimal therapeutic approach in many of the solid and hematologic cancers, including non-small lung cancer (NSCLC). Our study aimed to determine the costs of NGS assays for NSCLC diagnostics. Methods: We performed a micro-costing study of four NGS assays (Trusight Tumor 170 Kit (Illumina), Oncomine Focus (Thermo Fisher), QIAseq Targeted DNA Custom Panel and QIASeq Targeted RNAscan Custom Panel (Qiagen), and KAPA HyperPlus/SeqCap EZ (Roche)) at the StemCore Laboratories, the Ottawa Hospital, Canada. We used a time-and-motion approach to measure personnel time and a pre-defined questionnaire to collect resource utilization. The unit costs were based on market prices. The cost data were reported in 2019 Canadian dollars. Results: Based on a case throughput of 500 cases per year, the per-sample cost for TruSight Tumor 170 Kit, QIASeq Targeted DNA Custom Panel and QIASeq Targeted RNAscan Custom Panel, Oncomine Focus, and HyperPlus/SeqCap EZ were CAD 1778, CAD 599, CAD 1100 and CAD 1270, respectively. The key cost drivers were library preparation (34–60%) and sequencing (31–51%), followed by data analysis (6–13%) and administrative support (2–7%). Conclusions: Trusight Tumor 170 Kit was the most expensive NGS assay for NSCLC diagnostics; however, an economic evaluation is required to identify the most cost-effective NGS assay. Our study results could help inform decisions to select a robust platform for NSCLC diagnostics from fine needle aspirates, and future economic evaluations of the NGS platforms to guide treatment selections for NSCLC patients. Recent developments in next generation sequencing (NGS) technologies have revolutionized the field of genomics by providing the ability to perform massive parallel sequencing of large areas of the genome with high accuracy . The tecOur team at the Eastern Ontario Regional Laboratory Association and the Ottawa Hospital Research Institute conducted a research project to identify the best platform for NSCLC diagnostics, with the goal of developing a robust platform for NSCLC diagnostics from fine needle aspirates (FNAs). Although several commercial platforms exist for NSCLC-targeted diagnostics, none have undergone rigorous testing to assess their utility and associated costs in the clinical setting. The cost data are important and are relevant for resource allocation decisions at a population level. The existing studies show a substantial variation in the costs per patient of NGS testing in patients with cancer, ranging from USD 695 to USD 2861 ,13,14,15This study aimed to determine the per-sample cost of NGS assays in defining mutations in NSCLC molecular testing in Canada.We conducted a micro-costing study and estimated per-sample costs for four NGS assays commonly used for NSCLC from a provider’s (EORLA) perspective. These assays included the Trusight Tumor 170 Kit , the Oncomine Focus , the QIAseq Targeted DNA Custom Panel and the QIAseq Targeted RNAscan Custom Panel , and the SeqCap EZ/KAPA Hyper Prep Plus Custom . Micro-costing is a detailed costing approach in which all of the resources required for an intervention are identified and enumerated, and the unit costs are attached to these resources to estimate the total costs .In this study, the cost of each NGS assay included the costs of library preparation, sequencing, and bio-informatic analysis and interpretation. For each of the cost components, we identified the resources consumed during each activity and divided them into the following resource use categories: (1) personnel; (2) capital; and (3) supplies and reagents. The costs were reported in 2019 Canadian dollars.The data were collected at StemCore Laboratories at the Ottawa Hospital Research Institute over a 12-month period. We considered the resources required for the testing pathway, including capital, supplies and reagents, and personnel time to perform library preparation, sequencing, bio-informatic analysis, laboratory oversight, and training.We used a time-and-motion approach ,20 to meThe trained research assistant recorded the data pertaining to technical hands-on time to perform each SOP, using a stopwatch. The recorded time did not include overnight incubation periods and extended periods during which the samples were on equipment with minimal-to-no staff oversight. The data on the capital required for library preparation were collected through direct observation and a pre-defined questionnaire. The data were also collected on the equipment’s lifespan, maximum capacity, and current utilization. In addition, the questionnaire was used to record information on the supplies and reagents used for each step of the SOP to perform the testing. The personnel time required for laboratory oversight and attending training was recorded, using a data collection sheet.The data on the infrastructure required for bioinformatic analysis were collected through interviews with the bioinformatic staff, and included information on the software, storage requirements, and staff time required for data analysis, which covered time to set up an analysis pipeline for each assay, time for maintenance of an automated analysis pipeline, and time for data analysis.The acquisition costs for capital and the unit costs for supplies and reagents were based on market prices. The unit costs for supplies and reagents were derived by dividing their market price by the number of units contained within the item. Staff salaries were obtained from the budget reviews and were verified by the team. The midpoint of the salary ranges was used, and a working week was assumed to be 37.5 h and a working year was assumed to be 52 weeks. The costs of performing sequencing were obtained as lump-sum costs from the study team, and included the costs of required equipment and personnel time to perform sequencing.The cost for each assay was equal to a sum of the costs of personnel, equipment and consumables across library preparation, sequencing, bioinformatic analysis, and administrative support. We calculated the total costs as a product of the unit costs and resource use. We estimated the costs for DNA and RNA samples, except for SeqCap EZ/KAPA Hyper Prep Plus Custom in which only the cost for the DNA samples was reported, as the assay was not compatible with RNA.Guided by expert opinion, we assumed that the capital required for library preparation is comparable across the assays. For the costs of equipment, the acquisition cost for each unit was amortized over the lifespan of the unit at a discount rate of 1.5% per annum . The perThe costs for each assay were also categorized into fixed and variable costs. The fixed costs are those that do not vary by case throughput , while the variable costs are defined as costs that vary by case throughput . The per-sample cost for each assay was estimated by dividing the annualized costs by the annual laboratory throughput, which was based on the annual laboratory case throughput at the StemCore Laboratories. We performed a one-way sensitivity analysis to assess the uncertainty in the cost estimates by varying the annual case throughput and batch size between their respective upper and lower bounds, which were determined based on clinical expert opinion.The per-sample cost to process a DNA sample was CAD 1287.87 for the Trusight Tumor 170 Kit, CAD 1227.93 for the SeqCap EZ/KAPA Hyper Prep Plus Custom, CAD 1005.33 for the Oncomine Focus, and CAD 449.32 for the QIAseq Targeted DNA Custom Panel . The perOur study used a micro-costing approach to estimate the costs of four high-throughput genomic assays in NSCLC. The costs accounted for the personnel time, capital, and the supplies and reagents required for library preparation, sequencing, and bioinformatic analysis. Our study showed that the per-sample cost was highest for the Trusight Tumor 170 Kit , followed by the SeqCap EZ/KAPA Hyper Prep Plus Custom (DNA: CAD 1228), the Oncomine Focus , and the QIAseq Targeted DNA and RNAscan Custom Panels . It should be noted that each assay may have different performance characteristics and that the Trusight Tumor 170 Kit is the largest panel, which covers 170 tumor-related genes, meaning that the assay would allow the sequencing of many tumor genes, including those not yet approved for NSCLC diagnostics, without additional costs. However, the assay’s performance is beyond the scope of this study. Regardless of the type of assay, the NGS costs were primarily driven by the supply and reagent costs for library preparation (23–51%) and sequencing (32–52%). The fixed costs of capital accounted for only 5–10% of the total costs. These cost estimates were robust to changes in the case throughput and batch size .The costs of the NGS assays estimated in our study were within the range of costs reported in previous cost studies, which evaluated the cost of genomic sequencing procedures in NSCLC patients. Sabatini et al.  used a mOur study had some limitations that must be acknowledged. First, the data were collected from a single setting; our results may therefore not be generalizable to other settings, as the personnel time required to process a sample could vary across laboratories. The costs could change once the assays are moved into clinical practice and become part of the provincially funded system, due to additional costs relating to quality assurance protocols, expanded bio-informatics support, management, and oversight . Second,


Amidst the ongoing monkeypox outbreak, global awareness has been directed towards the prevention of viral transmission and case management, with the World Health Organization declaring the outbreak a public health emergency of international concern. Monkeypox virus is one of several species in the Orthopoxvirus genus, with other species of the genus including the variola, cowpox, mousepox, camelpox, raccoonpox, skunkpox, and volepox viruses. Although the nomenclature of these species is based on the animal host from which they were originally isolated, transmission from animals to humans has been reported with several species. The progression of disease, following an incubation period, typically consists of a prodromal phase with systemic flu-like symptoms. Various organ systems may be affected in addition to the formation of pathognomonic skin lesions. As monkeypox poses a continued public health concern, the ophthalmic sequelae of monkeypox virus, especially those leading to vision loss, warrant consideration as well. This review provides a comprehensive summary of the ophthalmic implications of poxviruses in clinical and laboratory settings reported in the literature, as well as areas of unmet need and future research. Poxviridae family. Several species are included in this genus, such as monkeypox virus and variola virus, the causative organism of smallpox. These species share similar characteristics, and many of these viruses have been shown to cause infection in humans, as seen in Orthopoxvirus (OPV) is a genus of complex, double-stranded DNA viruses of the There are many routes of transmission to the host, including parenteral, respiratory, and mucosal . The patThe diagnosis of OPV can be made through several methods. Electron microscopy of vesicle fluid or crusts is important in differentiating OPV from others, such as herpesvirus . HoweverThe aim of this review is to provide a comprehensive summary of the clinical manifestations, route of transmission, vaccines, treatments, and ocular implications of eight OPV species: monkeypox, variola, cowpox, mousepox, camelpox, raccoonpox, skunkpox, and volepox virus. We also discuss postulated mechanisms for recent zoonotic outbreaks and appropriate protective measures to prevent transmission.Monkeypox virus (MPXV) is a zoonotic OPV that was first identified and isolated in 1958 following the shipment of infected monkeys from Singapore to Denmark .MPXV can be transmitted through animal-to-human transmission through direct contact with blood, body fluid, and mucocutaneous lesions of an infected animal . Human-tSeveral studies have reported that vaccination against smallpox is about 85% effective in the induction of cross-protection against MPXV. A two-dose vaccine based on a modified attenuated vaccinia virus (Ankara vaccine) was authorized to prevent monkeypox ,18,19. TThere are currently no antiviral drugs explicitly approved for the treatment of MPXV infection. The antiviral cidofovir has rarely been used as an off-label treatment for MPXV in humans and, although reported to be efficacious, further clinical investigation is necessary ,21,22. TReports of ocular findings associated with MPXV infection include (a) periorbital, (b) conjunctival, and (c) corneal involvement. Individuals may report ocular redness, irritation, photosensitivity, discharge, and vision changes . The chaIn a study that investigated the potential reservoir competence of the Gambian pouched rat (GPR) for MPXV by utilizing a combination of in vivo and in vitro methods, whitish-yellow corneal opacities and ocular discharge were noted in the eye of the inoculated rats 14 days post-infection. High levels of viral shedding were also noted in the conjunctival/corneal swabs taken from one of the rats .Another study characterized a black-tailed prairie dog model of infection, featuring post-inoculation with the West African and Congo Basin strains of MPXV isolated during monkeypox outbreaks in 2003. Monkeypox viral DNA was detected in ocular swab samples 12 days post-infection, with periocular lesions also noticed 9–12 days post-infection in some animals .Variola minor virus was later identified in South Africa, the United States, and South America in the 19th century [Variola major, which was 5% to 25%, Variola minor was a less lethal form of the virus, with a CFP of <1% [Smallpox, caused by the variola virus (VARV), is described as one of the most lethal epidemic-causing diseases that resulted in an estimated 400 million deaths in the 20th century . Althoug century ,70,71. CP of <1% .Person-to-person transmission of VARV has been proven to occur through aerosols during close contact with infected persons ,73. It iThe variola vaccine was discovered in the late 18th century and helped achieve the global eradication of smallpox . The oriOcular findings associated with smallpox infection have been extensively documented and may involve anterior and posterior manifestations. As early as 1908, ocular involvement of smallpox was observed with (a) periorbital, (b) conjunctival, (c) corneal, and (d) posterior segment findings. Periorbital findings include rash, edema, and secretions. Conjunctival pathology consists of pustules and phlyctenules. Corneal involvement that presents as corneal ulceration is the most reported severe finding, with associated perforation with iris prolapse and hypopyon reported in some cases ,28,29. SAlthough rare, cases of ocular vaccinia, which refers to eye infections resulting from accidental autoinoculation of the virus from the immunization site to the eye, have been reported. Ocular vaccinia commonly involves the eyelid, with occasional conjunctival and corneal involvement, which may include stromal opacification and scarring ,31,32,33Cowpox (CPXV) is a rare zoonotic infection that can cause a self-limited disease, with more severe cases in the immunocompromised ,76. WhilThe true reservoirs of CPXV are now considered to be wild rodents rather than cows. Incidental hosts include cows, zoo animals, cats, and humans . CPXV trAn atypical clinical presentation of cowpox infection was reported in France in 2016. A smallpox-vaccinated patient developed thoracic varicella zoster-like-lesions following injury from a metallic guardrail previously stored in the ground. This occurrence stirred considerations for an alternative route of CPXV infection and the immunological status of smallpox-vaccinated patients for disease transmission .A case of fetal death following maternal CPXV infection was documented in France in 2017, highlighting the risk for complications associated with orthopoxvirus infection during pregnancy .Due to similarities between the two viruses, the smallpox vaccine provides cross-protective immunity against cowpox virus . CidofovOcular findings of cowpox include (a) periorbital, (b) conjunctival, and (c) corneal involvement. Reported cases of CPXV in humans include conjunctival chemosis and hyperemia progressing to keratitis and corneal ulceration, with severe cases requiring interventions such as amniotic membrane transplantation and autologous limbal stem cell transplantation ,43. AnotEctromelia virus (ECTV), also known as mousepox, closely resembles variola virus and monkeypox virus, two other members of the Orthopoxvirus genus: . These sNatural infection of ECTV is hypothesized to occur via microabrasions in the skin. The virus is speculated to initiate infection once it has reached the lower layers of the epidermis and dermis . The incVaccines and treatments specifically targeted toward ECTV eradication have not yet been closely studied in the literature.Ophthalmic symptoms of ECTV include conjunctivitis and blepharitis . The earCamelpox virus (CMLV) causes a smallpox-like illness in the camel . CamelpoTransmission of CMLV can occur directly via inhalation or through a skin abrasion . The virThe incubation period of CMLV is 9–13 days . The earBoth attenuated and inactivated forms of vaccination against CMLV are available for camels. Antiviral medications, most commonly cidofovir, can be used to treat infected animals and have a high level of efficacy .Skin lesions may develop on the eyelids. Retinal lesions have also been reported and may lead to blindness .Raccoons are the natural host of raccoonpox virus (RACV). There is little known about the epidemiology of RACV. However, RACV, along with skunkpox virus and volepox virus, is believed to be endemic to North America and, therefore, can be characterized as a “new world” orthopoxvirus .RACV was initially isolated from the respiratory tract of healthy-appearing raccoons in a swamp area in Maryland . In thisRACV is an effective vector for vaccine development due to its unique ability to trigger an immune response through mucosal routes . There aThere are no reported ocular complications associated with RACV infection.Skunkpox virus (SKPV) is rare and has been tentatively designated a member of the Orthopoxvirus genus . SKPV haLittle is known about the specific clinical manifestations of SKPV, as it has not been closely studied in the laboratory or the field.Like SKPV and RACV, volepox virus (VPXV) is categorized as one of the three New World or North American orthopoxviruses. It was first isolated from a scab of an otherwise healthy vole in California .The species of voles and mice in which VPXV is found have minimal contact with humans, so there is believed to be a low chance for transmission . VPXV haThere are no known vaccines or treatments specifically targeted toward VPXV eradication in the literature.Specific ocular findings include maculae and papulae occurring on the eyelids, as well as conjunctivitis .Measures can be taken to reduce the likelihood of monkeypox transmission in indoor settings. Hand hygiene should be performed after touching materials that have had contact with the monkeypox rash . InfecteRecommended PPE to be used by healthcare personnel includes a gown, gloves, goggles or face shield for eye protection, and a particulate respirator equipped with N95 filters or higher that has been approved by the National Institute for Occupational Safety and Health .A number of explanations for recent outbreaks have been proposed. These include individual immunologic phenomena in humans as well as behavioral considerations.Vaccination with the vaccinia virus has been shown to have cross-protective immunity against other orthopoxviruses such as VARV, MPXV, and CPXV . The speIn addition to proposed immunologic mechanisms for recent orthopoxvirus outbreaks, human behavior is believed to be responsible, in part, for recent orthopoxvirus infections in multiple ways. Although not fully elucidated, there is evidence indicating that physical contact and exposure to fluids of an infected animal may increase the risk of disease . A rise In particular, monkeypox transmission is closely associated with high-risk sexual behaviors. In the wake of the recent outbreak, a high proportion of monkeypox cases was noted in the men-who-have-sex-with-men (MSM) population ,115. OthAlthough there may be variation between strains, orthopoxviruses characteristically present with skin lesions with progression from papules to vesicles to scabs. Other associated symptoms may include fever, lymphadenopathy, malaise, and arthralgia. However, less is known about the ocular manifestations of these viruses. Findings may consist of eyelid lesions, conjunctival or corneal involvement, and posterior segment findings such as retinitis, with particularly severe cases leading to devastating consequences such as permanent vision loss.Outbreaks of zoonotic orthopoxvirus have occurred globally, the most notable being the ongoing monkeypox outbreak. Potential explanations for recent outbreaks include the population’s waning smallpox immunity and increased transportation of infected animals or fomites . Future


Rounded shapes, which have been shown to enhance sweetness, were compared to the perfectly symmetrical Platonic solids. In a first online experiment, participants were presented with a rotating three-dimensional geometric shape , and indicated their liking for the shape, as well as its perceived hardness, and its expected temperature. The sphere was liked best, followed by the Platonic solids. The sphere was also evaluated as softest, and received the warmest temperature ratings. By contrast, the Platonic solids were rated as harder and significantly colder than the sphere. Experiment 2 investigated whether the liked shapes were also evaluated as looking tastier. Ratings of expected tastiness and the appearance of five shapes selected based on high liking scores and fitted with edible and inedible visual textures were recorded. The sphere was rated as looking tastiest, with edible-textured rounded shapes resulting in significantly tastier ratings. Experiment 3 assessed the taste corresponding to each shape. A sweet and umami preference for rounded shapes was documented, with sour and bitter typically matched to angular shapes. Importantly, the Platonic solids were associated with several tastes. These findings are explained in terms of current theories of crossmodal correspondences, while considering how temperature and texture can be used to modulate expected liking. As one of the main criteria used in the visual identification of the objects in our environment, judgments of shape are implicated in almost every aspect of our daily lives. Furthermore, people typically prefer rounded versions/contours of objects, which are described as looking more beautiful. This preference for rounded shapes is not unique to humans, as it has also been documented in other species , and mayTimaeus. There are five Platonic solids , see perfectly symmetrical—no matter which direction it is turned/rotated. Importantly, the regular geometric shapes of the Platonic solids can be encountered in diverse natural aspects of everyday life, both animate  and inanimate . Plato considered them to be the building blocks of the natural world/universe, hinting, in their perfect symmetry, as having the potential to derive beauty from geometrical symmetry , or rather, the rounded shape effect will always take precedence as far as liking and aesthetic judgements are concerned. We approach this question by assessing people's liking of rounded versus Platonic shapes in a first study. We then take the Platonic solids that were most liked together with the sphere in a second study, where we test them crossmodally, by pairing the various geometric shapes with edible and inedible textures. Our third study assesses the basic taste that corresponds to each of the nine geometric shapes used in the three experiments reported here.Geometrical symmetry allows for transformations in any part of an object, while the object in its entirety remains unchanged, a property also described as ondences . Their sondences . In thisAs such, in Experiment 1, the participants were presented with a selection of geometric shapes including the five Platonic solids and a sphere, two irregular geometric objects custom-built such that they appeared as having an irregular angular shape and an irregular rounded shape  had a mean age of 23 years . Participants were recruited from socializing apps  and gave their informed consent to take part in the study. The study was approved by the Ethics Board of the Alexandru Ioan Cuza University of Iasi, Romania (no. 2325/24.05.2021). This study conforms to the Declaration of Helsinki  together with a visual analog scale (VAS). The participants rated the presented shape on the given VAS, with only the end anchors visible. The VASs used were set from 0 to 100 points, where 0—lack of the measured variable, 50—moderate presence, and 100—maximum recorded. Note that the VAS provides an effective means of capturing characteristics that vary along a continuum which cannot always be measured directly. They also provide an excellent means of capturing participants’ subjective response along a measured scale see .The manipulated independent variable was Geometric shape . Each geometric shape was presented until a response was detected, for each of the three VASs recorded as dependent measures: Liking (very unpleasant – very pleasant), Hardness (very soft – very hard), and Temperature (very cold – very hot). Four repetitions were recorded for each visual stimulus. In total, each participant received 108 trials in randomized order. The experiment lasted for between 15 and 20 min.Q–Q plots), together with skewness and kurtosis calculations. Following the previous literature , to investigate the rounded-shape effect in 3D, the Liking scale data were consequently analyzed with a one-way repeated-measures analysis of variance (ANOVA) with the factor Geometric shape . The Liking ANOVA was followed-up with a set of post-hoc hypothesis-driven tests in order to investigate the existence of a rounded-shape effect, by comparing the sphere, with all of the irregular control shapes.The data from each of the three VASs  were inspected visually with histograms and sample/theoretical quantiles . Note that the irregular angular/rounded shapes were not used in these analyses, because no hypothesis existed with respect to the Platonic solids as compared to the Bouba-like/Kiki-like shapes. These ANOVAs were followed-up by paired-samples  t-tests . Statist t-tests .Liking: The results indicated a significant main effect of Rounded shape . Specifically following-up the rounded-shape effect, the results indicated that the sphere  was liked significantly more than both the irregular angular shape , and the naturalistic irregular shape . Moreover, when comparing all of the geometric shapes, a significant main effect of Geometric shape was evidenced , with the sphere once again significantly more liked than either the cube  or the tetrahedron . From the Platonic solids, the octahedron  was liked significantly more than the naturalistic irregular control shape . None of the other post-hoc t-tests survived the familywise-error correction , indicating that the Platonic solids were rated as looking significantly harder  was perceived as being significantly softer than the cube , the tetrahedron , the octahedron , the icosahedron , and the dodecahedron . A very similar pattern of results was also observed for the naturalistic irregular control shape , which was perceived as being significantly softer than the cube , the tetrahedron , the octahedron , the icosahedron , and the dodecahedron . Importantly, the sphere did not differ significantly in terms of its expected hardness from the naturalistic irregular shape (p = n.s.).rder see . The sphTemperature: The results of Experiment 1 indicated a significant main effect of Temperature ; see M = 48.93; SE = 4.30) was rated as significantly warmer than the cube , the tetrahedron , and the neutral control shape . In this case, the geometric naturalistic irregular shape was rated as looking significantly colder than the octahedron .The results of Experiment 1 confirm that the sphere is the most liked of the geometric shapes. The additional scale results reinforce the notion that the sphere was rated significantly different in terms of its hardness and temperature: The sphere is visually perceived as being softer and also thermally moderately warm. As far as the Platonic solids are concerned, we find significant differences in all three of the recorded measures. The results highlight, quite unexpectedly, that geometric shapes closest to the sphere, such as the custom-built rounded irregular shape, the icosahedron, and the dodecahedron, but also, in certain respects the naturalistic irregular control shape, received similar ratings as far as liking, hardness, and temperature are concerned. This result could be indicative of a general effect of a rounded shape giving rise to increased liking cf. . Having Temperature assessments are all situated below/on the moderate demarcation of the scale used in our study. Interestingly, the results of Experiment 1 indicate that assessments of temperature for the geometric shapes that were presented are generally aligned with those of liking, thus indicating that rounded shapes are evaluated as warmer, with the angular geometric shapes being thermally evaluated as closer to the cold end of the VAS. Here, it is worth mentioning that certain of the Platonic solids  were evaluated as being very similar to the sphere , apparently indicating a symmetry effect as far as geometric shape – temperature correspondences are concerned.Experiment 1 was designed to assess regular versus irregular geometric shapes with respect to pleasantness, together with other sensorial qualities, such as expected hardness and temperature. The geometric shapes were presented in a neutral grey color, so as not to induce any implicit taste–color correspondence; it has been demonstrated that people tend to associate the texture of whitish cotton candy with the taste of sweetness . In Expea priori power calculation with an α = .05 and 1−β = .80 indicated that a sample size of 60 participants was necessary to detect an effect of f = .25 in the data , each fitted with one of the edible  and inedible textures . All of the images had the same height (9.3 cm) and width (17.3 cm), with the contained object set to a height of 7 cm/6°40′ degrees of visual angle, when assuming a standard distance of 60 cm from monitor. The 3D stimuli were rotated with the in-built turntable Blender function. Six 2D stimuli depicted the individual textures of the experiment  and Texture . The dependent variables were two 0–100 VASs for Tastiness  and Appearance (very smooth – very rough). Each participant completed a total of 120 experimental trials. At the end of the experiment, the six 2D shapes were presented in a randomized order, together with two buttons (Edible and Inedible) and the participants were required to make a choice according to what they thought each texture represented.t-tests.Averages of the data collected for the two VASs were analyzed with repeated-measures ANOVAs with the factors Geometric shape  and Texture , followed by Holm-corrected Tastiness: The results indicated a significant main effect of Geometric shape  on tastiness ratings. The sphere  was rated as significantly tastier than the icosahedron , the dodecahedron , the octahedron , and the naturalistic irregular control shape . The naturalistic control shape looked significantly less tasty than the Platonic dodecahedron .F = 75.21; p < .001; 2pη = .511) was also evident on tastiness ratings, as well as an interaction between Geometric shape and Texture  were rated as looking significantly tastier than the inedible shapes , as expected. None of the hypothesis-driven post-hoc t-tests performed following the two-way interaction  survived the correction for multiple comparisons.A main effect of Texture  on the appearance data. The sphere  was rated as looking significantly smoother than all of the Platonic solids tested in Experiment 2, including the icosahedron , the dodecahedron , the octahedron , and the naturalistic irregular control shape . On the other hand, the naturalistic irregular control shape was rated as looking significantly rougher than the Platonic octahedron , the icosahedron , and the dodecahedron .F = 18.39; p < .001; 2pη = .203) in the appearance data. Post-hoc tests indicated that the chocolate texture  was rated as looking significantly smoother than the cookie texture , sand , and grass . Furthermore, the cheese texture  appeared significantly smoother than the sand , and grass textures . The cookie texture was rated as looking rougher than the wood , but smoother than the sand texture . Lastly, amongst the inedible textures, wood was rated as being significantly smoother than both sand  and grass . Averaging over all edible/inedible shapes’ appearance data, and comparing them with paired-sampled t-tests revealed that the edible shapes appeared overall significantly smoother , than the inedible shapes .There was a significant main effect of Texture . The remaining sample  had a mean age of 26 years old .An a priori power calculation with an The apparatus and materials were the same as in Experiment 1. The participants were informed that the experiment was designed to investigate any links between basic tastes and geometric shapes, were given a short introduction to the fifth basic taste – umami, for anyone who was unfamiliar with it , after wFor each of the geometric shapes, we calculated frequency tables of the five basic tastes and we then used Pearson's Chi-squared test to compare the observed frequencies in our sample to the taste frequencies predicted by chance. For the analysis, we considered equal predicted frequencies for each of the basic tastes in each of the nine geometric shapes tested in Experiment 1 .x-axis from the naturalistic irregular shape on the left, through the rather angular shapes of the irregular angular shape and Platonic tetrahedron, octahedron, and the cube, up to the rounded shapes of the irregular rounded shape, the Platonic icosahedron and dodecahedron, with the sphere positioned furthermost on the right of 2[4] = 138.43; p < .001), some exhibiting two tastes chosen at above-chance levels , or even three . Another visually striking pattern in the data is the change in associated taste, evident through the clustering of sour and bitter frequencies for the angular shapes depicted on the left of The results of Experiment 3 demonstrate that our participants tended to pair the rounded shapes with the tastes of sweet and umami, whereas the more irregular and angular shapes were associated with the basic tastes of sourness and bitterness instead. It should be noted that these results are in line with classic studies on shape-taste associations  as well We present the results of three online studies designed to investigate symmetry in judgments of liking for various rounded as opposed to angular shapes. To this end, our main interest was in evaluating the perfectly symmetrical Platonic solids, a set of five regular polyhedra first described by Plato. These are regular shapes that occur in nature and because they are perfectly symmetrical, in a rotational sense, it has been suggested that their structure is derived from symmetry and in turn, reflects beauty . Because so it is unsurprising that our control rounded shape was also significantly paired with the sweet taste.The sphere was liked best of all and also, along with our custom-made irregular rounded shape, was rated as appearing warmest and softest, amongst all of the geometric shapes tested in Experiment 1. It has been demonstrated that context-free geometric shapes can act as emotional primitives and thus they can convey emotion, with pointy downward-facing V's perceived as threatening, as opposed to their rounded counterparts, which are rated as pleasant . The samThe Platonic solids, on the other hand, were rated as looking significantly harder and were also associated with a lower temperature. Hardness is a clear characteristic of Platonic solids, as they have all received above-moderate ratings. The hardness of the Platonic shapes is not necessarily anchored in symmetry, but rather, seems to act as a counter to the rounded shape. Angular in our study thus relates to a cooler temperature, hardness, and also to roughness of texture. To follow the Platonic shapes’ implied beauty, the Gestalt principles of harmony, contrast, and dissonance have been put forward as drivers of flavor perception , all theexpected liking or taste, as opposed to actually experiencing the taste, hardness, or temperature of a certain geometric shape, such as this would happen when sampling foodstuffs in the real-world needs to be acknowledged (e.g., see the crossmodal correspondence elicited between sound and temperature, by both imagining and perceiving water of different temperatures, Because the results of the three experiments presented in this study are based on expected evaluations of liking, hardness, temperature, tastiness, and appearance/texture, when interpreting the present results the distinction between evaluating the Of note, the results of Experiment 3 indicate that some shapes, especially amongst the regular Platonic solids, are not strongly associated with a particular basic taste. For the cube, which was rated as looking hardest, because of its regularity and angularity, its pairing with the bitter taste was expected. Quite unexpectedly, the cube is also paired with the sweet taste. In this match, perhaps dominant would be the learned association, both semantic, as well as visual, with a sugar cube. By contrast, the rounded shapes were expected to set a significant expectation of sweetness, and this was indeed confirmed. However, it is evident from the results of Experiment 3 that rounded symmetrical shapes also correspond to the taste of umami. The color-taste correspondence has been studied intensely, nevertheless, the umami taste hasn’t been, until now, consistently matched with a certain shape, even though it seems that when designers create shapes/colors for specific tastes, they are indeed likely to use a rounded shape .Such a taste-versatility as found for the Platonic solids begs the question of how this can be used crossmodally? Could we perhaps top the rounded shape dominance in aesthetic judgments so long as we use hardness, temperature, and/or texture more friendly or fitted to a Platonic solid? It would certainly be interesting to pair the shape of a Platonic solid with another sensorial attribute to elicit a specific desired effect, in the culinary field, but also in design and other creative artistic domains (e.g., see To consider the implications of the research presented in this manuscript, recent years have seen growing interest in the putative mechanisms underlying the crossmodal correspondences, and their relation with other kinds of crossmodal mapping, such as semantic correspondences and amodal relations . Under tTaken together, our results confirm the dominance of the rounded shape as far as liking is concerned. The liked rounded shapes, together with the Platonic solids tested carry a taste for beauty, as not only are they most liked, but also evaluated as tastiest. Our investigation of rotational symmetry over three experiments further extends the current knowledge, by highlighting that enhanced liking of rounded shapes is supported by thermally warmer evaluations. Importantly, angular symmetrical shapes are perceived as harder and colder in terms of their expected temperature. Future research needs to capitalize on these novel sensorial qualities that define symmetrical angular geometric shapes—dynamically assessed in rotational motion as in the present study—while at the same time considering the potential offered by the symmetrical geometrical shapes taste versatility.


Scientific progress in understanding, preventing, treating, and managing viral infections and associated diseases exemplifies the extent to which research on small DNA tumor viruses has impacted human health. Various diagnostic tools for detecting virus infections and effective prophylactic vaccines against human papillomavirus (HPV) and hepatitis B virus (HBV) now promise to reduce the disease burden of these viruses in the coming decades. At the same time, these small DNA viruses, including the polyomavirus SV40 and adenoviruses, have been indispensable tools for studying molecular mechanisms of carcinogenesis and basic cellular functions. Fundamental processes of cellular biology, such as transcriptional regulation, splicing, and DNA damage, have been elucidated, at least in part, through the study of the replication cycles of such viruses and their interactions with the host. Nowadays, they also serve as valuable tools outside the research setting as viral vectors in numerous gene therapies and vaccines. Despite the advent of new technologies, research on small DNA viruses remains relevant to discovering novel biological mechanisms and clinical applications further to reduce disease burden. In this Special Issue, we have collected work that showcases current and ongoing work towards these goals.Four studies tackle unanswered questions related to diseases caused by small DNA viruses. Sasivimolrattana et al. provide evidence that the virome in HPV-infected tissues is associated with HPV status and abundance using a metagenomic analysis . As the Two articles explore the basic questions in biology of two small DNA viruses: papillomavirus and adenovirus. Moreno et al. demonstrate evidence in support of the long-standing hypothesis that infected tissue progenitors  are preferential targets for papillomavirus-induced transformation, using the preclinical murine papillomavirus infection model . Their rIn addition to the original research articles, this Special Issue includes four review articles to update recent progress in small DNA virus research. Suresh and Menne summarize recent preclinical studies of developing antivirals, immunomodulators, and therapeutic vaccines for chronic hepatitis B, using the Eastern woodchuck with woodchuck hepatitis virus as an HBV laboratory animal model . Loke etFor several decades, our studies in small DNA viruses have pioneered breakthroughs in understanding fundamental molecular and cellular mechanisms. The articles published in this Special Issue demonstrate that this process continues. Yet, new questions and challenges to eradicating the diseases caused by small DNA viruses do not seem to be disappearing. While discovering detailed views of virus–host interactions and pathogenesis, we comprehend the extreme complexity of virus–host interactions and pathogenesis interweaved with other microbes and host immunity as an ecological system. In the host cell, the virus manipulates various cellular processes not fully understood, such as epigenetic regulations, noncoding RNAs and RNA modifications, posttranslational mechanisms, and metabolic pathways. In the clinic, there is an urgent need to develop novel antivirals, prophylactic and therapeutic vaccines, and biomarkers to effectively diagnose and treat patients with persistent virus infection and virus-associated diseases. Working together, we will continue towards achieving our ultimate goal of controlling virus infection and virus-associated diseases. Finally, we thank all authors for contributing to this Special Issue.


While no Kunjin virus was detected in these mosquitoes, we applied our viral replicative intermediates screening system termed monoclonal antibodies to viral RNA intermediates in cells or MAVRIC to this set of samples. This resulted in the detection of 28 pools with virus replicating in C6/36 mosquito cells and the identification of three insect viruses from three distinct virus classes. We demonstrate the persistence of the insect-specific flavivirus Palm Creek virus in Coquillettidia xanthogaster mosquitoes from Darwin over almost a decade, with limited genetic drift. We also detected a novel Hubei macula-like virus 3 strain in samples from two mosquito genera, suggesting the virus, for which the sequence was originally detected in spiders and soybean thrips, might be involved in a horizontal transmission cycle between arthropods and plants. Overall, these data demonstrate the strength of the optimized MAVRIC system and contribute to our general knowledge of the mosquito virome and insect viruses.Mosquitoes ( Aedes aegypti , which is only found in Queensland [Culex annulirostris , Ae. vigilax  and Coquilletidia xanthogaster  [KUN), as well as the alphaviruses Ross River virus, Barmah Forest virus and Sindbis virus [Surveillance of the prevalence of arthropod-borne viruses in mosquitoes is key to the control of arthropod-borne diseases in humans and animals in Australia. There are over a hundred mosquito species in the Northern Territory (NT), which include all the major potential and actual mosquito-borne disease vectors in Australia, except for 2, around two crocodile farms in the Darwin region of the NT. The aim of this surveillance was initially to monitor the WNVKUN distribution in mosquitoes of this region as it had been detected in farmed crocodiles of the area [In this context, mosquitoes were collected, using encephalitis virus surveillance (EVS) traps baited with COthe area ,4,5.KUN high risk period (January to July) [Cx. annulirostris and one Cx. quinquefasciatus ) and the 4879 remaining mosquitoes were identified as females. The majority of the mosquitoes collected were Cx. annulirostris (62%) followed by Mansonia uniformis (12%) , Anopheles bancroftii (7%) , Cx. palpalis (6%) , Cx. pullus (4%) , Cq. xanthogaster (2%), Cx. quinquefasciatus (2%) and 14 other species; see Between three and six overnight traps were set at various locations within two crocodile farms in rural areas of the Darwin region in the NT. Traps were set in the afternoon and collected the following morning after sunrise, fortnightly between the 15 March and the 27 April 2018. This corresponds to the middle of the peak arbovirus activity period (between January and May) and of the WNVl-glutamine, with two glass beads following a cycle of 3 min at 30 Hz in a Tissue Lyser III . The homogenates were clarified by centrifugation at 12,000× g for 10 min then filtered through a 0.8/0.2 µm sterile filter. The filtered homogenates were inoculated in four wells of a 96-well plate pre-seeded with C6/36 mosquito cells in 5% FBS RPMI with the same additives  as above and left to incubate for five days at 28 °C. These cells were selected to maximize the chances of detecting all mosquito-borne viruses present in the samples. After incubation, the supernatants were harvested and passaged in a similar fashion for three passages and the leftover supernatants stored at −80 °C. The inoculated cells were fixed in either 20% acetone in phosphate buffered saline  with 0.02% bovine serum albumin for two hours at 4 °C or 4% formaldehyde in PBS with 0.5% TritonX-100 for ten minutes at 4 °C. We performed a fixed-cell ELISA as described previously using pan-flavivirus monoclonal antibodies (mAbs) 4G2 [KUN replication, so using vertebrate cells instead may have led to some WNVKUN detections. However, this experimental design enabled the identification of 28 pools (14.14%) with replicating virus, positive for dsRNA in the MAVRIC ELISA after three passages on C6/36 cells.To maintain the required cold chain for virus isolation work, mosquito traps were transported from site in an insulated container with dry ice before mosquitoes were stored at −80 °C. Mosquito identification to species level was performed on cold tables at the Medical Entomology laboratory in Darwin using taxonomic keys. Identified mosquito species were stored in vials up to 50 specimens. Mosquito pools were shipped to the laboratory on dry ice and then stored at −80 °C until processing for virus isolation. The mosquito pools were homogenized in 2 mL tubes in Roswell Park Memorial Institute (RPMI) medium with 2% fetal bovine serum (FBS), 50 U penicillin/mL, 50 μg streptomycin/mL and 2 mM Aedes birnavirus (mAb A7E6) [Alphamesonivirus-4 Casuarina virus (mAb C.5D3) [Alphamesonivirus-1 Nam Dinh virus (mAb N.4H7) in three Culex pools, with a prevalence of 1.52% (3/198 pools), see These 28 samples were selected for further identification, first using a panel of insect-specific virus mAbs, developed in our laboratory, to screen for viruses known to circulate in Australian mosquito populations. We found that none of the samples contained the seadornavirus Liao ning virus (mAb 6E6) , negevirAb A7E6)  or Alphab C.5D3) . HoweverCq. xanthogaster mosquitoes. The RT-PCR products were gel purified using the Nucleospin gel and PCR clean up kit  following the manufacturer’s instructions and subjected to sequencing by the Sanger method at the Australian Genome Research Facility .In total, 10 of the remaining 25 unidentified MAVRIC-positive samples had inconsistent MAVRIC-binding profiles depending on the fixative used, and were suspected to be insect-specific flaviviruses, undetected by flavivirus mAbs 4G2 and 4G4; see O’Brien et al., 2021 for details on this peculiar binding profile . We therCq. xanthogaster mosquitoes collected in Darwin in 2010 [Cq. xanthogaster mosquitoes collected from Kununurra, Western Australia, in 2010 [Cq. xanthogaster, the only species in which PCV has been detected so far. The calculated minimum infection rate in this mosquito species in our samples was 6.82% (6/88 Cq. xanthogaster).All sequenced samples were identified as Palm Creek virus (PCV)  using Geneious Prime . The calculated percentage identities for nucleotide and amino acid are displayed in Cx. annulirostris) and extracted RNA from the third passage supernatant using the Nucleospin RNA Virus isolation kit , following the manufacturer’s protocol—without carrier RNA. This extract was then deep sequenced as follows. RNA was treated with Heat & Run DNase  to remove contaminating host DNA, and the DNAse was heat inactivated at 80 °C for five minutes. First strand cDNA was generated using Protoscript II , using the supplied random primer mix and reaction conditions as recommended by the manufacturer. The reaction product was then converted to double-stranded DNA (16 °C for 60 min) using an enzyme mixture consisting of E. coli DNA ligase, DNA polymerase I and RNase H , followed by heat inactivation (80 °C for 5 min). The double-stranded cDNA was used as template for library construction using the Nextera XT library kit  with barcoded primers. The library was sequenced on a NextSeq 500 generating 2× 150 bp paired reads.We selected one sample from the remaining 19 unidentified MAVRIC-positive pools, DS16  using the blastn algorithm on NCBI BLAST [E-value 0.0): Pyongtaek Culex Macula-like virus [Cx. bitaeniorhynchus  mosquitoes collected in South Korea in 2018.De novo assembly of the data yielded 10 million reads, 6.5M of which were assembled in a 5914 nucleotide-long genome sequence, which was identified as a macula-like virus (Genbank accession number MW959137). It was most closely related to the three published sequences for Hubei macula-like virus 3 (HMLV3)  with 77–79% nucleotide identity over 64–93% of the query cover . The overall nucleotide sequence alignment showed that our strain of HMLV3 shared 77% identity with the three published HMLV3 strains and 69% identity with Pyongtaek Culex Macula-like virus see . Over thnder 85% . These dAn. bancroftii) was identified as a second isolate of our HMLV3 strain, bringing the prevalence of this virus in our samples to 1.01% (2/198 pools) . The primer pair amplifies a 486 base pair amplicon in the 5′ region of the genome and was used with the SuperScript III One-Step RT-PCR System with Platinum Taq DNA Polymerase  with a melting temperature of 50 °C and a 30-second extension time. One of the tested samples  see . Amplicoe family ,22,23,24There are still 17 samples that remain MAVRIC positive and unidentified. These may contain new virus species within the families tested here, or novel viruses from insect virus families that have gone undetected in Australian mosquitoes so far. High throughput sequencing of RNA derived from these samples could help identify these unknown replicating viruses.Cq. xanthogaster mosquitoes from the Darwin area over almost a decade, with limited genetic drift. We also detected a novel Hubei macula-like virus 3 strain in samples from two mosquito genera, suggesting the virus, originally detected in spiders and soybean thrips, might be involved in a horizontal transmission cycle between arthropods and plants. Overall, these data contribute to our general knowledge of the mosquito virome and insect viruses.We have shown that human disease-centered mosquito surveillance can yield data on the mosquito virome, when using the optimized MAVRIC detection system . We were