Saccharomyces boulardii by ionic gelation using agavins (A) and whey protein (WP) as wall materials and to evaluate the morphostructural changes that occur during in vitro gastrointestinal digestion. Encapsulations at different levels of A and WP were analyzed using microscopic, spectroscopic and thermal techniques. Encapsulation efficiency and cell viability were evaluated. S. boulardii encapsulated at 5% A: 3.75% WP (AWB6) showed 88.5% cell survival after the simulated gastrointestinal digestion; the bead showed a significantly different microstructure from the controls. The mixture of A and WP increased in the survival of S. boulardii respect to those encapsulated with alginate, A or WP alone. The binary material mixture simultaneously allowed a controlled release of S. boulardii by mostly diffusive Fickian mechanisms and swelling. The cell-release time was found to control the increment of the Damköhler number when A and WP were substrates for S. boulardii, in this way allowing greater protection against gastrointestinal conditions. Encapsulation is a process in which a base material is encapsulated in a wall material that can protect it against external factors and/or improve its bioavailability. Among the different encapsulation techniques, ionic gelation stands out as being useful for thermolabile compounds. The aim of this work was to encapsulate  Among the compounds that can form this wall material can be found polysaccharides, lipids, proteins and their mixtures ,12. DiffThe morphostructure and the release mechanism of the capsules can be modified to suit specific applications and release sites of bioactive compounds and/or cells . MorphosIn the study of the release of bioactive compounds/cells from the encapsulating matrix, three stages must be considered: (a) release from the surface, (b) diffusion through the swollen matrix and (c) erosion of the matrix . KineticThe Zero-Order model (Equation (1)) considers the released fraction to be independent of the initial concentration ,20:Mt/M∞t, amount released after time t; M∞, amount released at infinity or equilibrium, k, rate constant. Fraction released independent of initial concentration. Remarks: MHiguchi’s model (Equation (2)) is applicable to diffusion-controlled release through water-filled pores and can be affected by pH and temperature ,21,22:MtKorsmeyer–Peppas model (Equation (3)) applies when the system has more than one release mechanism :Mt/M∞ = Remarks: For spherical particles, n ≤ 0.43 for Fickian diffusion (Case I transport); 0.43 ≤ n ≤ 0.85 for non-Fickian (diffusion or swelling); n ≥ 0.85 for a Case II transport; and n > 1 for a Super Case II transport.Finally, the Peppas–Sahlin equation (Equation (4)) quantifies the relative contributions to the Fickian and relaxation transport ,24:Mt/M∞d tm for diffusion and kr t2m for Case II transport.Remarks: kMost mathematical modeling studies have been used only in pharmaceutical studies . Regardiα-lactoalbumin, which represent approximately 70% of total proteins and are responsible for the hydration, gelling and surfactant properties of protein ingredients [Enterococcus faecium, Lactobacillus acidophilus, Bifidobacterium pseudocatenulatum CECT 7765, Bifidobacterium bifidium and Saccharomyces boulardii. The mentioned materials improve the encapsulation rate, viability during storage and simulated gastrointestinal testing, producing more stable beads with potential application in different foods, as they significantly preserve the quality of food matrices [6–107 CFU g−1 at the time of consumption [Saccharomyces boulardii is a yeast considered a probiotic, offering protection against antibiotic-induced diarrhea, ulcerative colitis and Crohn’s disease [w/w), as well as to the human body temperature of 37 °C. This microorganism is usually dispensed in lyophilized form in soft gelatin beads, while its use in food matrix processing is scarce [S. boulardii as a model, which used agavins as a carbon source, where a greater growth of the population density was observed when such compounds were added to the culture medium [S. boulardii using different techniques  seemed to have potential as an oral delivery system in pharmaceutical or food applications. In turn, it benefits both the survival and the bioavailability of S. boulardii within beads, making it more effective in the prevention and treatment of gastrointestinal diseases [S. boulardii capsules obtained by ionic gelation using the response surface methodology.Wall materials such as whey protein (WP) and reserve polysaccharides such as agavins (A) have been shown to be favorable materials for the encapsulation of probiotic microorganisms and bioactive compounds ,27,28,29redients . Severalmatrices ,41,42. Tmatrices  defines sumption  and thatsumption . Sacchar disease . It is rs scarce . The prediseases ,27,48. Adiseases . The stu®, molecular weight 216 g/mol, purity (95–100%). Whey protein was purchased from General Nutrition Center  with the following components: protein (80%), ash (5%), lactose (4%) and fat (4%). Lyophilized yeast cells were obtained from Floratil Saccharomyces boulardii CNCM-I 745 beads . Yeast extract on peptone dextrose agar (YPD agar) was obtained from Sigma-Aldrich Química S. de R.L. de C.V., containing agar (10 g/L), bacteriological peptone (20 g/L), glucose (20 g/L) and yeast extract (10 g/L).The agavins were obtained in powder form following the Mexican patent MX/a/2015/016512 (Modular system and process for obtaining different products from agave fructans). Sodium Alginate REASOLSaccharomyces boulardii cells were reactivated three times; they were first poured into 50 mL of sterile YPD broth and incubated for 24 h at 37 °C. Subsequently, a flask containing 50 mL of YPD broth was inoculated with the resulting medium at 5% and incubated for 12 h at 37 °C. Finally, a flask with 30 mL of YPD broth was inoculated with 2% of the resulting medium and incubated for 12 h at 37 °C [Lyophilized at 37 °C . The lasDispersions of agavins and whey protein were made by mixing the powders in distilled water at neutral pH and room temperature (25 ± 2 °C) and stirring for 30 min with a magnetic stirrer until complete dissolution. Both dispersions were stored for 24 h at 4 °C to achieve complete hydration. Dropwise addition of 1 M NaOH or 1 M HCl solution was used to obtain samples at the required pH range ,50.Simultaneously, 30 mL solution of 0.96% alginate was prepared in a 50 mL Erlenmeyer flask. Then, the dispersions of agavins, whey protein and the total cell concentrate recovered from 30 mL of YPD broth were carefully added. Everything was mixed with a magnetic stirrer at 25 ± 2 °C until complete homogenization.2-factorial design was used, with three replicates for each of the nine resulting treatments  under sterile conditions [To determine the concentrations of the wall materials of the capsules, a completely randomized experimental 3eatments , taking l design . Encapsunditions ,53.t = detected concentration of the incorporated material (CFU), and Wi = initial concentration (CFU).The viability of yeast cells was quantified by %EE, which is defined as the detected concentration of the incorporated material in the formulation with respect to the initial concentration used . It was Threshold function. Those were manually adjusted to a grayscale range. Using these bead images, size (area and perimeter) and shape (circularity and solidity) parameters were analyzed. Morphostructural parameters of size and shape were evaluated in order to identify which beads were significantly influenced by the effect of different concentrations of wall materials (agavins and whey protein). A response surface analysis was then performed for the parameters described above, using the Minitab Graphs software (Minitab® 18.1).Micrographs were obtained from wet beads using a stereo optical microscope , a fixed digital camera  and MetaMorph Software . Thirty micrographs (with reflected light and 3X magnification) were obtained for each one of the five types of beads that showed a significant difference in terms of %EE and were stored in color *.tiff format. Those micrographs were subjected to digital image analysis using ImageJ software (v1.53s). The bead images were converted to grayscale (8 bits) and binarized (black and white) using the S. boulardii inside the wet beads, a laser scanning confocal microscope (Carl Zeiss Model CLSM 800) with Smartsem 5.6 software was used to obtain the micrographs. Two fluorochromes were used: propidium iodide and acridine orange. Five micrographs were taken on the central zone of each bead type  and were stored in color *.tiff format. They were subjected to digital image analysis using ImageJ software (v1.53s). Using the Color Threshold function, images were manually adjusted to a color scale to specifically select yeasts. The yeast images were binarized, and the following parameters were obtained using the GLCM (gray level co-occurrence matrix) texture plug-in: second angular momentum, contrast, correlation and entropy. Subsequently, the images were skeletonized using the Skeletonize tool. Fractal dimension and lacunarity parameters were obtained from these images using the Frac Lac plug-in. This skeletonization was performed based on the distribution of S. boulardii in the bead, since it has been observed that the cells tend to deposit in the crosslink structure of the wall materials [Beads Internal Networking (BIN) or mesostructure, formed both individually and in combination by the alginate, agavins and whey protein.To evaluate the distribution of aterials ,56. ThisBeads External Surface (BES), were observed using a Carl Zeiss EVO LS 10 scanning electron microscope  with Zeiss Efficient Navigation Software (2.3 Blue Edition) at 40X and 500X optical magnification with backscattered electrons in the environmental mode. Five micrographs were taken for each type of bead (40X magnification). Meanwhile, micrograph sections were taken in different areas  of the beads and were stored in color *.tiff format. Micrographs were subjected to a digital image analysis using ImageJ software (v1.53s). The different micrograph sections were converted to grayscale (8 bits). Based on these, the following parameters were analyzed using the GLCM texture plug-in: second angular momentum, inverse differential momentum, contrast, correlation and entropy. Subsequently, the SDBC  plug-in was used to obtain the value of the parameter texture fractal dimension.The external morphology and texture of wet beads, called −1 in the range of 400–4500 cm−1. The determinations were performed in triplicate.Thermal profiles were obtained using differential scanning calorimetry equipment  under a nitrogen atmosphere. Each sample was heated from 0 to 300 °C at a rate of 5 °C/min. All determinations were made in triplicate. The equipment was preliminarily calibrated with an indium standard reference . Fourier−1. First, 100 mg of beads were taken, and 10−1 to 10−5 dilutions were made in 1.5 mL Eppendorf tubes with 900 μL of 0.9% Sodium Citrate Sigma-Aldrich®, molecular weight 294.10 g/mol (mixing homogeneously with a vortex until complete dissolution of the beads). Subsequently, 10−3–10−5 dilutions were seeded in Petri dishes (mixing homogeneously with a vortex) using the microdroplet method proposed by Miles and Misra [The different capsules were subjected to human gastric simulation according to the INFOGEST protocol  under thnd Misra . They we−1) into the medium were quantified using a spectrophotometer at a wavelength of 640 nm, while viable cell counts were quantified according to the plate count method on YPD agar and measured as CFU mL−1. The release of yeast cells was analyzed according to the Higuchi (Equation (2)), Korsmeyer–Peppas (Equation (3)) and Peppas–Sahlin (Equation (4)) models. In addition, the parameters in each equation were determined. Finally, the Damköhler number (Da) was determined by Equation (6):Five grams of beads were weighed and added in SSF, SGF and SIF release solutions . The con® 18.1), IBM SPSS Statistics (Version 25.0) and Microsoft Excel version 16.62 (22061100) software were used for statistical analysis. One-way ANOVA  and Tukey’s multiple-comparison test (α < 0.05) were performed. For data analysis and illustration, the Minitab Graphs package (MinitabS. boulardii was carried out in relation to time (h) and optical density (O.D.). It was observed that the peak of the logarithmic phase occurred at 12 h with an O.D. of 1.34 [S. boulardii cells to reach a concentration of 106 CFU mL−1 and, therefore, the time required to proceed to perform the encapsulation process.The growth kinetics of  of 1.34 . This waThe bead with the highest %EE was AWB9 with 96.77%, while the AWB7 beads had the lowest efficiency of 89.27%. The bead types that had a significant difference compared to controls were: AWB3 (96.043%), AWB5 (93.901%), AWB6 (95.698%), AWB8 (94.053%) and AWB9 (96.775%). These capsules were used for subsequent analyses . S. boulardii compared to whey protein, due to the fact that beads with intermediate and high percentages  of agavins had the highest %EE.Therefore, agavins had a significant influence on the protection of area , perimeter , circularity (measure of how close the bead shape is to a perfect circle) and solidity (the ratio between the bead area and the convex hull of the bead).Morphostructural parameters were analyzed by stereo microscopy. According to Pedreschi et al. , the strarea and perimeter) were significantly affected (circularity and solidity) parameters with different concentrations of wall materials was observed using a response surface analysis  were obtained from concentrations of approximately 3.4% whey protein and 4% agavins  from concentrations of 3.8%, while whey protein presented high solidity values from concentrations of 4.8%.  while delimiting a smaller amount of space (perimeter) with circularity and maximum solidity (the structure should be as solid as possible), it was desired that the bead shape be as similar as possible to a circle. Therefore, a mathematical optimization of these parameters was carried out for the AWB3, AWB5, AWB6, AWB8 and AWB9 beads. Area, circularity and solidity were weighted as maximizing them, while minimizing was set as a target for perimeter, and the degree of importance was set homogeneously. As a result of the mathematical optimization, three types of beads were obtained that met or were close to the previously established objectives: AWB5 (3.75% A: 3.75% WP), AWB8 (3.75% A: 5% WP) and AWB6 (5% A: 3.75% WP).In order to contain the greatest number of cells , contrast , correlation  and entropy (a measure of disorder in the BIN) [fractal dimension  and lacunarity  were also assessed. Hristu et al. [GLCM texture plug-in provide information about the spatial relationships between pixel intensities in the Beads Internal Networking (BIN).the BIN) ,16,63. Msecond angular momentum, contrast, correlation and entropy, AWB5, which had equal concentrations of agavins and whey protein (3.75% A: 3.75% WP), showed a significant difference only compared to B and WB, but not AB. Therefore, it was deduced that agavins predominated these parameters, i.e., as they had a significant effect on the uniformity, variability, correlation and the disorder of the internal structure of the beads compared to B and WB. In the combination of agavins and whey protein wall materials, the complexity  of the agavins predominated since the AWB5 was significantly different from B and WB. Thus, the agavins significantly influenced the complexity of the bead structure. The lacunarity presented a significant difference for AWB5 with respect to AB and, at the same time, similarity with B and WB. Therefore, for this parameter, it was the whey protein that had a significant influence. In summary, the agavins had a direct effect on the parameters of second angular momentum, contrast, correlation, entropy and fractal dimension. This means that agavins determined the internal structure of the beads compared to the whey protein.According to the parameters  second asecond angular momentum, inverse differential momentum , contrast, correlation, entropy and SDBC  [BES   were evare) [BES . The seccorrelation, the AWB5 and AWB6 beads significantly presented the highest values of correlation, compared to B, AB, WB and AWB8. For entropy, the beads that presented a significant difference with the lowest disorder value were AWB5 and AWB6 compared to all other bead types. The B, AB and WB beads did not present significant differences among them. The AWB5 bead presented a significant difference with the lowest tortuosity value compared to AB. Because it was found in equal concentrations in the wall material, this suggested that the whey protein governed the SDBC parameter. In general, the whey protein determined the external structure of the beads.Regarding −1 assigned to C-O-C stretching groups and peaks at 1400 and 1600 cm−1 corresponding to asymmetric and symmetric stretching of COO- bonds. As for the band observed at 3250 cm−1, this was attributed to O-H stretching groups [−1 was identified. Santiago-García, et al. [−1 for agavins-type fructans. Particularly, the 900–1200 cm−1 absorption bands in the carbohydrate region were attributed to the vibrations of the C-O-C group in the cyclic structures, indicating a significant carbohydrate content [−1 derived from the N-H bending vibrations of the amide bond and C-N stretching [−1, so there was similarity with the WB bead, since it had a peak from 1570–1700 cm−1, a consequence of the C=O vibrations of peptide bonds and the direct relationship they possess with the secondary structural elements: β-sheets, α-helices, chain turns and random coils belonging to its main proteins, β-lactoglobulin and α-lactalbumin [g groups ,66,67. R, et al.  describe content ,70. The retching ,71,72. Ktalbumin ,71,72.−1 peak was present in AB, attributed to the vibration of the C≡N group , and the 2800–2990 cm−1 peak was a consequence of the symmetric stretching of the CH3 and CH2-O groups, where the latter peak was also found in WB [The FT-IR spectra  were ablnd in WB . The correlation coefficients (R) of the spectra were analyzed, where it was observed that AWB6 beads showed a higher correlation with AB (0.9620). In turn, AWB8 beads showed a higher correlation with WB (0.9572).The thermograms of the beads produced by the alginate, agavins and whey protein, either individually or in combination, are shown in AWB6 presented an endothermic peak at 155.82 °C , which wpure wall materials (with S. boulardii). The fusion temperature of the AWB5 bead was 138.860 °C , while the AWB6 bead was 155.820 °C , and finally, the AWB8 bead was 149.360 °C .The AWB5, AWB6 and AWB8 beads presented a shift of the endothermic peak due to the combination of alginate, agavins and whey protein in different concentrations, forming mixtures that provided properties different from those of the −1), while the images represent the viable/dead cells at specific points (at the beginning and at the end) of in vitro gastrointestinal digestion. The results of this assay were verified by viability tests.Micrographs of the different capsules  at the bAccording to the results obtained with the plate count technique, the AWB6 beads (5% A: 3.75% WP) showed the highest %viability (88.539) compared to B (70.37%), AB (75.58%) and WB (72.66%), while the AWB5 (83.99%), AWB6 and AWB8 (86.26%) beads did not significantly differ from each other.S. boulardii release was related to the concentration of yeast detected in the medium and the amount of live yeast inside the bead  obtained, which allowed us to know to what extent the regression line best represented the model [2 values (0.8538–0.9848) in all cases, so it was selected as the model that best described the release and transport of S. boulardii in the different types of beads. The B, AB, WB and AWB5 beads presented relaxation release mechanisms. In this case, the polymeric chains of the alginate changed their configuration during the intestinal phase and thus led to the release of the microorganisms. The AWB6 bead (5% A: 3.75% WP ) had the least % cell release  governed these systems. This means that they contributed significantly to the trapping of available S. boulardii in the bead. In contrast, the controls had values Da < 1, indicating that the rate of transport governed the system, i.e., more yeast was diffused from the system than was generated [For the Damköhler number (Da), the AWB5, AWB6 and AWB8 beads presented values Da > 1, thus showing that for these beads, the reaction rate  compared to the controls  . AgavinsOn the other hand, whey protein provided a smoother homogeneous external structure, with less contact surface and optimal size that ensured the highest number of microorganisms were encapsulated in the smallest space. Moreover, whey protein did not compromise the fracture of the internal/external microstructure of the bead, due to the cohesiveness that globular-type proteins impart to the network ,80. Through DSC and FT-IR analysis, it was found that the mixture of agavins and whey protein maintained and/or compensated the weak interactions that allowed increases in the melting temperature and the energy needed to generate a change in the properties of the compounds that formed the bead. 7 CFU/mL) after in vitro gastrointestinal digestion compared to AWB5, AWB8 and the controls, thus ensuring that it reached the concentration necessary to be considered functional [The optimal type of mixture for encapsulation was AWB6 (5% A: 3.75% WP), which presented the highest %viability  had a Fickian diffusion transport mechanism, probably as it used the concentration gradient as the driving force with a release mechanism controlled by the bead microstructure . The mat2, perimeter: 9.082 μm), and induced a Fickian release mechanism with both transport phenomena, diffusive and relaxation, simultaneously (mainly diffusive) according to the Peppas–Sahlin model [The optimal agavins/whey protein mixture obtained capsules with a more crosslinked matrix. This may be due to its gelling strength developed by agavins and whey protein in an alginate matrix, which enhanced the hardness and flexibility of capsules  . The use of mixtures of agavins and whey protein using ion gelation increased the survival of


We conducted a genomic analysis of monkeypox virus sequences collected early in the 2022 outbreak, during July–August , in Washington, USA. Using 109 viral genomes, we found low overall genetic diversity, multiple introductions into the state, ongoing community transmission, and potential for co-infections by multiple strains. The World Health Organization declared the 2022 mpox (formerly monkeypox) outbreak a public health emergency of international concern on July 23, 2022, after cases were identified in nearly 80 countries  can augment contact tracing efforts and identify emerging variants, which potentially could affect infectivity, virulence, vaccine escape, and treatment resistance. By late August 2022, Washington had deposited more monkeypox virus (MPXV) sequences into public databases than any other state in the country. Here, we describe the Washington outbreak by using 109 MPXV genomes collected in the state.https://github.com/greninger-lab/revica), a custom pipeline that performs trimming, filtering, and iterative remapping  value <31 (range 15.9–30.4). We performed sequencing by using a hybridization probe-capture–based approach, as previously described . We geneemapping . Sequencemapping  Table. Wemapping . This stThe analysis comprised a total of 109 sequences from 98 persons whose specimens were collected during July 6–August 19, 2022, primarily from King and Pierce Counties. Of the 98 patients, 90 (91.84%) were male and 1 (1.02%) female; 7 (7.14%) had unknown or undeclared sex. Median age at specimen collection was 36.0 (range 19–57) years.We identified multiple identical genomes from different persons, suggesting ongoing community transmission , panel AOverall, we observed low genetic diversity and a median of 1 aa (range 0–7 aa) mutation (substitutions or deletions) across the genome relative to the B.1 ancestor . We identified 138 unique SNPs across the genome in the 109 sequences , panel BSequences from multiple swabs from the same person at the same time point had a median pairwise nucleotide difference of 1 (range 0–10 for 11 sample pairs) outside of labile tandem repeat regions  Table.Overall, our data showed ongoing community MPXV transmission in Washington. The limited MPXV genetic diversity makes it challenging to use WGS data for contact tracing. However, continued genomic surveillance will be crucial for tracking viral evolution and identifying mutations associated with vaccine escape or antiviral treatment resistance.Additional information on genomic analysis of early mpox virus outbreak strains, Washington, United States.


Virus discovery has been fueled by new technologies ever since the first viruses were discovered at the end of the 19th century. Starting with mechanical devices that provided evidence for virus presence in sick hosts, virus discovery gradually transitioned into a sequence-based scientific discipline, which, nowadays, can characterize virus identity and explore viral diversity at an unprecedented resolution and depth. Sequencing technologies are now being used routinely and at ever-increasing scales, producing an avalanche of novel viral sequences found in a multitude of organisms and environments. In this perspective article, we argue that virus discovery has started to undergo another transformation prompted by the emergence of new approaches that are sequence data-centered and primarily computational, setting them apart from previous technology-driven innovations. The data-driven virus discovery approach is largely uncoupled from the collection and processing of biological samples, and exploits the availability of massive amounts of publicly and freely accessible data from sequencing archives. We discuss open challenges to be solved in order to unlock the full potential of data-driven virus discovery, and we highlight the benefits it can bring to classical (mostly molecular) virology and molecular biology in general. The way viruses are identified has changed fundamentally since the first viruses were discovered at the end of the 19th century due to technological advancements. The first discovery of a virus, now known as tobacco mosaic virus, by Dimitri Ivanovsky in 1892 . The disIrrespective of the technology used, researchers aiming to discover novel viruses were dependent on the availability or collection of biological samples and their analysis in wet labs, the latter involving DNA/RNA extraction, library preparation, and sequencing ,10,11,12An advantage of DDVD over conventional virus discovery approaches that cannot be overestimated in our view is the amount of available data, which outcompetes by a large margin that of any conventional virus discovery data set. As of June 2022, NCBI’s SRA (and its mirrors at the European Bioinformatics Institute (EBI) and the DNA Data Bank of Japan (DDBJ)) included data for more than 10.4 million sequencing experiments conducted on 8.6 million different biological samples. About 8.3 million of the experiments were on eukaryotes, and the large majority of the data sets (90.7%) were publicly accessible, while the remaining <10% were under controlled access. Although about 3.2 million experiments were of human origin, the SRA contained data for about 119,000 different species, with 70.8% of them being eukaryotes. The accumulation of new sequencing experiments in the SRA during recent years has proceeded at a rate that strongly exceeds linear growth, with a size increase of 21.4% just within the last year and a current doubling period for the amount of open access data of approximately two years . This inNotably, it has been demonstrated that the vast amount of unprocessed data in sequencing archives can be analyzed efficiently through highly parallelized computation using high-performance computing ,24 or clConventional virus discovery studies typically look for viruses (i) in sick hosts, focusing on predefined pathogenic viruses or virus groups, (ii) in economically important hosts, or (iii) in often poorly characterized hosts living in a geographically well-defined area, for instance, at the human–wildlife interface of a certain country or state. The discovery of viruses via DDVD in samples collected for other purposes can offer a fresh perspective for virus diversity research and beyond. For instance, the uncontrolled  presence of viruses may account for unexplained variance in the outcome of experiments of the same kind involving a certain host by different laboratories or the same laboratory over a longer period of time. DDVD enables the identification of such missing variables and, therefore, offers a way to connect to researchers of various expertise outside virology. Taking this reasoning another step forward, DDVD could stimulate the field to develop standards that enable the identification and (genetic) characterization of all organisms in a biological sample, including bacteria and other microorganisms, even if only incomplete sequences can be obtained, which could, nevertheless, provide valuable information ,27.The availability of often very detailed metadata presents another notable strength of DDVD. In the case of the SRA, information about the organism (at the species or genus taxonomic rank) from which the sequencing sample was obtained, as well as tissue or even cell type annotation, is typically available. This offers the opportunity to assign the host and possibly organ tropism of the newly discovered viruses. Moreover, we expect that the confidence of host assignment, at a reasonable and useful taxonomic rank, can be further increased by taking into account viral phylogeny, as closely related viruses generally tend to have similar hosts, particularly in the case of viruses infecting eukaryotes . Host asAdditional opportunities of DDVD that should not be overlooked relate to the data it relies on. Firstly, we emphasize that the usage of these data for scientific purposes is free of financial costs beyond standard investments in IT infrastructure that any laboratory has to make. The whole scientific community has already spent billions of USD (conservatively assuming an average cost of at least 100 USD per sequencing experiment) to generate the data in the SRA and similar repositories, which can now be re-used freely and accessed publicly. The analysis of the data will also not induce additional costs if scientific high-performance computers are utilized, or assuming that charges normally made by cloud-computing providers can be circumvented . Second,In order to realize the full potential of DDVD and maximize its impact on virology, several main issues require the development of a community-wide consensus. We believe that it will be prudent to generate and apply quality standards for viral genome assemblies produced by DDVD studies to ensure their reliability and acceptance by the virology community. We consider this to be of high relevance because of the exceptionally large number of assembled viral sequences that have already been produced and will be produced in future studies, making comprehensive manual curation unfeasible. Such quality standards should be able to identify chimeric sequences produced by mis-assembly, incomplete sequences , and other sources of error, such as PCR/sequencing artifacts, each constituting a large challenge for both conventional metagenomics-based virus discovery and DDVD. Ideally, quality control of viral genome sequences and fragments assembled in DDVD studies will advance current standards  and may Another challenge that DDVD has in common with many conventional metagenomics-based virus discovery studies is brought by the considerable fraction of viral genome sequences that are not coding-complete ,24,25. FThe relatively large number of incomplete genome sequences produced by DDVD studies highlights the need to develop advanced and tailored sequence assembly tools. Specifically, such assemblers would be able to cope with strongly varying and locally poor read coverage, as this constitutes a major reason for fragmented assembly results. Another challenge associated with viral genome assembly, particularly in the case of RNA viruses, is an extraordinarily high sequence variability that is regularly observed at hypervariable regions of viral genomes, which usually cannot be resolved by standard sequence assemblers. In our experience, manual intervention during the assembly process can help to overcome such coverage gaps and variability hotspots of viral quasispecies divergence, suggesting that it may be feasible to design new algorithms for improved automated viral genome assembly. For instance, assembling based on the encoded protein sequence  would beBesides generating complete viral genome sequences, it will be equally important to further advance the sensitivity of detecting highly divergent viruses. The identification of viral sequences in sequencing data typically relies on the availability of related viral reference genomes and the ability of computational tools to discriminate between similarity due to homology from chance similarity or convergence ,23,24,40The advent of large-scale DDVD studies poses the question of whether the community is in need of new paradigms for publishing viral sequences discovered in silico by analyzing sequencing archives. We encourage the community to discuss the value and significance of the product generated, i.e., the assembled viral sequences, in light of the fact that the raw data have been generated by others. It is without question that the authors of the original studies in which the data were produced must be properly cited and acknowledged, which also includes linking between respective database entries. Likewise, it is, in our opinion, equally important to recognize that DDVD researchers generate new knowledge from these data that would, in all likelihood, have remained hidden otherwise. Many of the newly assembled viral sequences are not present in any public database, except in the form of raw reads in the respective sequencing repository. We, therefore, argue that it would be counterproductive to designate DDVD results as being secondary data. Currently, DDVD researchers are confronted with certain hurdles when publishing their results, for instance, during submission to NCBI/GenBank. It is typically required to submit the viral genome sequences to a special database division called Third Party Annotation (TPA), seemingly downgrading their status to some extent. As the massive inflow of viral sequences from DDVD studies will by no means stop in the foreseeable future, we encourage the community to discuss whether this approach is still timely.An even more pressing question, in our opinion, relates to the type of evidence that should be required for accepting the discovery of a bona fide virus. This concerns viral replication competence and particle formation, but also the packaging of different genome segments in the case of segmented and multipartite viruses. Classical virology would request to demonstrate each of these activities by respective wet-lab experiments. DDVD researchers, however, typically do not have access to the original specimens, and data submitters usually do not store their samples for longer periods of time, making canonical “biological validation” an unreachable goal in the vast majority of cases. We, therefore, encourage the virological community to (re)consider comparative genomics and phylogenetics as providing sufficient evidence for viral presence and identity. Assuming adequate sequence assembly quality (see above), the detection of homologous viral protein domains, segregated in a specific order, in an assembled sequence via sequence comparison with often well-characterized reference viruses makes it highly unlikely that the discovered sequence is anything else than of viral origin. Properties, for instance the enzymatic activity of viral polymerases, including conserved catalytic residues, can thus be accurately inferred for the newly discovered virus (genome) by transferring functional annotations from experimentally characterized reference viruses. Adding secondary or tertiary protein structure information, which can now be computationally predicted with high accuracy , to the Another important problem is posed by viral sequences integrated into eukaryotic host genomes, often termed endogenous viral elements (EVEs). These can often be discriminated from genomes of extant viruses by flanking host sequences and the accumulation of nonsense and frameshift mutations ,50,51,52Comparative genomics and phylogenetics, therefore, provide scalable and accurate tools that can cope with the constantly increasing number of sequences from DDVD studies and can be used to validate and characterize this invaluable source of new knowledge.We observe that public repositories of (unprocessed) Next-Generation Sequencing (NGS) data are growing at a more–than-linear rate, with the vast majority of NGS projects being unrelated to virological research questions. If the specimen subjected to NGS was infected by a virus at the time of sampling, viral genomes will be sequenced as unrecognized by-catch.The abovementioned developments prompted the emergence of Data-Driven Virus Discovery (DDVD), which is not primarily technology-driven, but rather data-centered and computational, is uncoupled from the collection and processing of biological samples, exploits publicly and freely accessible data from sequencing archives in high-throughput, and utilizes cluster- or cloud-based high-performance computing. Furthermore, DDVD requires the development of community-wide quality standards, highlights the need for tailored methods and approaches for viral sequence assembly, homology detection, and host assignment, and emphasizes the value of partial viral genome sequences. DDVD also challenges paradigms for required evidence of virus presence and identity, and demands for discussing adjusted (sequence) publication policies.DDVD brings benefits for virology and molecular biology as it offers a promising avenue toward a possibly comprehensive description of the virosphere, can connect in silico and wet-lab functional research in virology, offers a way to address the uncontrolled presence of viruses in a sample to account for unexplained variance in the outcome of experiments in molecular biology, and can be generalized to all biological entities in a sample, including bacteria, other microorganisms, and other selfish genetic elements.


The Oncotype DX© 21-gene Recurrence Score (RS) estimates the risk of distant disease recurrence in early-stage estrogen receptor-positive, human epidermal growth factor receptor-2-negative (ER+/HER2− ) breast cancer. Using RS to estimate risk of locoregional recurrence (LRR) is less conclusive. We aimed to perform network meta-analysis (NMA) evaluating the RS in estimating LRR in ER+/HER2− breast cancer.A NMA was performed according to PRISMA-NMA guidelines. Analysis was performed using R packages and Shiny.P < 0.050).16 studies with 21,037 patients were included (mean age: 55.1 years (range: 22–96)). The mean RS was 17.1 and mean follow-up was 66.4 months. Using traditional RS cut-offs, 49.7% of patients had RS < 18 (3944/7935), 33.8% had RS 18–30 (2680/7935), and 16.5% had RS > 30 (1311/7935). Patients with RS 18–30 (risk ratio (RR): 1.76, 95% confidence interval (CI): 1.32–2.37) and RS > 30  were significantly more likely to experience LRR than those with RS < 18. Using TAILORx cut-offs, 16.2% of patients had RS < 11 , 65.8% had RS 11–25 , and 18.0% with RS > 30 . LRR rates were similar for patients with RS 11–25 ; however, those with RS > 25 had an increased risk of LRR  compared to those with RS < 11. There was a stepwise increase in LRR rates when applying traditional and TAILORx cut-offs (both RS testing accurately estimates LRR risk for patients being treated for early-stage ER+/HER2− breast cancer. Future prospective, randomized studies may validate the predictive value of RS in estimating LRR.The online version contains supplementary material available at 10.1007/s10549-022-06580-w. Breast cancer is the most frequently diagnosed malignancy in women, with a lifetime risk of 12.4% in the western world , 4. MoreWithin the setting of early-stage estrogen receptor-positive, human epidermal growth factor receptor-2-negative (ER+/HER2− ) breast cancer, the Oncotype DX© Recurrence Score (RS) 21-gene expression assay  is used to predict distant disease recurrence following treatment with curative intent and to estimate prognosis , 9. ThisWhile the 21-gene expression assay is useful in gauging the potential benefit of prescribing cytotoxic chemotherapy in high-risk cases, its utility in estimating LRR risk is less apparent: Tumor blocks retrieved from the NSABP-B14 and B20 trials were evaluated with the sole purpose of establishing the risk of distant disease recurrence in a subset of ER+/HER2− lymph node-negative (LN-) breast cancer patients, before being combined to develop the RS signature . The preA systematic review was conducted in accordance to the ‘Preferred Reporting Items for Systematic Reviews and Meta-Analyses’ (or PRISMA) extension statement for reporting of systematic reviews incorporating network meta-analyses of healthcare interventions . Local iAll published studies with full-text manuscripts comparing LRR rates per RS category following treatment with curative intent for early-stage ER+/HER2− breast cancers were included. Randomized controlled trials and observational studies of a prospective and retrospective design were included. Included studies were expected to report on the primary outcome of interest. All studies failing to fulfill the pre-determined inclusion criteria were excluded. Conference abstracts, case reports, case series with less than 5 patients, editorial articles, opinion pieces, and review articles were excluded. Our rationale for omitting such articles was due to the paucity of crude data available in such forms for inclusion in meta-analysis. Studies not published in the English language were excluded. Included studies were not restricted by year of publication.Using the PICO framework , the aspPopulation–Patients who had previously been diagnosed with an invasive ER+/HER2− breast cancer aged 18 years or older who had undergone RS testing on their resected specimen from time of diagnosis.Intervention/Exposure–Any patient who subsequently developed LRR during follow-up (which includes those who have developed LRR with or without distant disease recurrence).Comparison/Control–Any patient free of LRR during follow-up.LRR rates for each RS category , using a pre-determined search strategy that was designed by the senior author (MJK). This search included the search terms: [(21-gene assay) OR (oncotype)] AND  linked using the Boolean operator ‘AND.’ Manual cross-referencing of reference lists from previous studies was undertaken.Manual removal of duplicate studies was performed before all titles were screened. Thereafter, studies considered to be appropriate had their abstracts and/or full text reviewed. Retrieved studies were reviewed to ensure inclusion criteria were met for the primary outcome, with discordances in opinion arbitrated through consultation with a third author (JPOD). Data extraction was also performed by two independent reviewers (MGD & EFC), with study details, basic patient clinicopathological characteristics, RS data, LRR rates, surgical data, and adjuvant treatment strategies all recorded. The final search was performed on the 22nd October 2021.2) tests as appropriate). Rates of LRR for each RS group were expressed as dichotomous or binary outcomes, reported as odds ratios (ORs) and risk ratios (RRs) were expressed with 95% confidence intervals (CIs). ORs/RRs were calculated, using crude event study data, to compare interventions using per-protocol data, where applicable. The lowest RS cut-off category  were used as the principal comparator for all analyses.Descriptive statistics were used to outline characteristics of included studies (Fisher’s Exact (†) and Chi-Squared Overall, there were data included from 21,037 patients with mean age at diagnosis of 55.1 years (range: 22–96 years). The mean RS was 17.1 (range: 0–71) and the mean follow-up was 66.4 months (range: 27.0–120.0 months). In total, 2.8% of included patients experienced LRR .In total, 64.0% of patients underwent breast-conserving surgery (BCS)  and 33.1% underwent mastectomy . Of those reporting treatment with adjuvant radiotherapy (XRT), 99.2% of patients eligible for BCS received breast XRT (5297/5339–11 studies). Following RS testing, 43.5% of patients received combined chemoendocrine therapy . The majority of patients underwent endocrine therapies .P < 0.001, χ2). Additionally, LRR rates increased when comparing dichotomous RS cut-offs: RS < 18: 2.2% vs. RS > 18: 6.0%  and RS < 30: 2.7% vs. RS > 30: 10.2%  , 33.8% had RS 18–30 2680/7935), and 16.5% had RS > 30 (1311/7935). Alternatively, 52.0% of patients had RS > 18 (4269/8213) and 83.5% of patients had RS < 30 (6610/7921)    and RS > 30  were significantly more likely to experience LRR than those with RS < 18 . LRR rates increased using dichotomous cut-offs: RS < 11: 1.2% vs. RS ≥ 11: 2.0%  and RS < 25: 1.7% vs. RS ≥ 25: 2.9%  , 65.8% had RS 11–25 , and 18.0% with RS > 30 . Alternatively, 83.1% of patients had tumors with RS > 11  and 84.3% had RS < 30     and RS > 25  compared to those with RS < 11 . While these findings are similar to the LRR rates observed in the seminal NSABP-B14 and B-20 trials at 10-year follow-up , this comparison is limited by the shorter duration of mean follow-up in the current analysis . Additionally, we acknowledge this is not a matched comparison due to our inclusion of LN + disease in the current study. Of note, the data outlined in the study by Mamousas et al. contribute a large proportion to the LRR rates observed in each of the RS groups delineated using traditional cut-offs, which is best explained through their inclusion of node-positive cancers only (based on the inclusion criteria of NSABP B28)  and increased again when applying dichotomous cut-offs . In our NMA, the relative risk of LRR was similar for RS < 11 versus RS 11–25, however was 2.41 times more likely in those with RS > 30. Furthermore, those with lower RS were significantly less likely to experience LRR at meta-analysis . However, caution must be taken when interpreting these results. The inclusion of data from the prospective TAILORx trial represents a large proportion of the patients evaluated using TAILORx cut-offs , which inevitably heavily influences LRR outcomes for this cohort [When applying the TAILORx cut-offs, this stepwise increase in LRR rates remained consistent [RS < 11: 1.2%, vs. RS 11–25: 1.8%, vs. RS > 25: 2.9% . While these previous studies evaluated LRR risk by receptor status, this NMA has the advantage of solely quantifying LRR risk using the 21-gene expression assay in ER+/HER2− disease, highlighting the value of genomic testing in estimating LRR rates in this subgroup of breast carcinoma.The current NMA outlines the risk of LRR based on results of the 21-gene expression assay in ER+/HER2− disease. As outlined by Goldhirsch et al. at the 2013 St. Gallen International Expert Consensus , genomicThe present study has several limitations. Firstly, and most importantly, the studies failed to provide data to allow accurate subgroup analysis in relation to LRR adjusted for age, menopause status, margin positivity levels, chemotherapy prescription, and most importantly, surgical management and XRT use. Therefore, conclusions drawn as to how to best apply RS testing to influence locoregional control of the breast and axilla may be perceived to be somewhat limited. Secondly, data from the prospective TAILORx trial represent a large proportion of the patients evaluated in this NMA using TAILORx cut-offs (69.3%), which heavily influences these outcomes in relation to LRR. Similarly, the patients included in this study by Sparano et al. represent 46.2% of the total patients included . Thirdly, just 37.5% of the included studies were of a prospective design (6/16), indicating most studies were retrospective, observational studies which are inherently subjected to ascertainment, confounding, and selection biases. Finally, publication dates of included studies ranged from 2010 to 2021, during which period the seminal results of TAILORx and RxPONDER have transformed the management paradigm for patients diagnosed with early-stage ER+/HER2− breast cancers. Despite these limitations, the current study is the first systematic review to provide real-world data estimating the risk of LRR based on results of the results of genomic testing for early-stage breast carcinoma. Moreover, this analysis contributes to current efforts focused on expanding indications for the 21-gene expression assay in the modern breast cancer treatment paradigm –38.In conclusion, RS testing accurately estimates the risk of LRR for patients being treated with curative intent for early-stage ER+/HER2− breast cancer. While RS testing is validated for quantifying the risk of distant disease recurrence, awareness for its ability to predict LRR is essential to establish effective locoregional control of the breast and axilla. Future prospective, randomized studies may further validate the predictive value of RS in estimating LRR and the application of RS to establish adequate locoregional control in high-risk cases.Supplementary file1 (DOCX 303 KB)Below is the link to the electronic supplementary material.


The study aims to describe the lingual laser frenotomy perioperative protocol for newborns with ankyloglossia with or without breastfeeding difficulties developed by Odontostomatology and Neonatology and Neonatal Intensive Care Units of the Aldo Moro University of Bari.Authors carried out a prospective observational cohort study. Newborns with ankyloglossia (classified by using both Coryllos’ and Hazelbaker’s criteria) with or without difficult breastfeeding (according to Infant Breastfeeding Assessment Tool) underwent diode laser frenotomy  and follow-up visits after seven and thirty days postoperatively. The authors analyzed as main outcomes the perioperative pain intensity measured by the C.R.I.E.S. scale, the occurrence of complications and quality of healing, the quality of breastfeeding, newborn’s postoperative weight gain, maternal nipple pain, and the presence of lesions as secondary outcomes.p < .05).Fifty-six newborns were included in the current study. Intraoperative mean pain intensity was 5.7 ± 0.5 points, resolved within thirty postoperative minutes. Observed complications were mild punctuating bleeding, carbonization of the irradiated site, and transitory restlessness. All wounds were completely healed within the thirtieth postoperative day. During follow-up, a significant breastfeeding improvement was evident with satisfactory newborns’ weight gain and a significant reduction of nipple pain and lesions (Our lingual laser frenotomy protocol provided significant breastfeeding improvement in the mother-newborn dyads with low intraoperative pain and no significant complications. Ankyloglossia, also known as “tongue-tie”, is a congenital anomaly meaning short lingual frenulum limiting mobility and impairing tongue function ; anyway,p < 0.05.A prospective observational cohort study was carried out in compliance with the principles of the World Medical Association Declaration of Helsinki on medical research protocols and ethics and the Aldo Moro University of Bari institutional review board (IRB) approved the study protocol , developed by the Unit of Neonatology and NICU and the Unit of Odontostomatology. From September 2020 to September 2021, infants were included according to the following criteria: ankyloglossia according to Coryllos’ criteria with the indication for lingual frenotomy as determined by the Hazelbaker et al.’s Assessment Tool for Lingual Frenulum Function (ATLFF)  and newbp < 0.001) and decreased significantly both immediately after frenotomy  and thirty minutes later . During frenotomy, the main complication  was punctiform bleeding, while carbonization of the irradiated site occurred in 11 newborns (19.6%). No injuries to Wharton salivary ducts were recorded. At the seventh-day follow-up visit, one mother (1.8%) reported punctiform bleeding due to accidental trauma on the surgical wound that occurred during the first application of the protective gel. No infections at the site of frenotomy (both wound and surrounding tissues) were found. Refusal of the pacifier was reported in 39 cases (69.6%) and 15 (26.8%) were frequently awake as unexpected postoperative complications, anyway, none of the participating parents needed for administering acetaminophen to the infants for managing such signs of discomfort. During the thirtieth-day follow-up visit, newborns’ behavior was reported by all mothers to be not different from the preoperative period. Figure p = 0.001). Newborns’ weight variation is shown in Fig. p < 0.001 and p = 0.001 respectively). Before surgery, 12 mothers (21.4%) had painful nipple lesions ; seven days after laser frenotomy, 10 (17.9%) still reported lesions, while only 2 (3.6%) at thirty days. Mean nipple pain was 4.3 ± 1.6 points before surgery, significantly reduced to 2.5 ± 2.2 points seven days after surgery (p < 0.001), and further reduced to 1.1 ± 1.1 points (p = 0.006) thirty days after surgery  because parents refused the laser procedure due to the absence of breastfeeding difficulties. Therefore, 56 newborns were included: 30 males (53.6%) and 26 females (46.4%). 34 (60.7%) were exclusively breastfed, and 22 (39.3%) received infant formula too. The mean age at frenotomy was 47.2 ± 20.2 days. According to Coryllos’ classification system, the cases of anterior ankyloglossia were 10 newborns (17.9%) with type 1 tongue-tie and 18 (32.1%) with type 2, whereas the cases of posterior ankyloglossia were 26 (46.4%) with type 3 tongue-tie and 2 (3.6%) with type 4. Their mean ATLFF function score was 7.8 ± 1.9 points whereas their mean appearance score was 4.8 ± 2.0 points. Their preoperative mean IBFAT score was 8.6 ± 1.9 points, and 24 (42.9%) had scores ≤ 8. None of the included patients showed lip-tie in addition to tongue-tie. All newborns completed the thirty-day follow-up. The analysis of pain intensity over time measured by using the C.R.I.E.S. scale is shown in Fig. 2, and erbium are used, but diode lasers are widely used for the surgery of oral soft tissues, also for their relative costs [The incidence of ankyloglossia ranges from 0.02% to 11.00% , 14–16 ave costs , 23. Tisve costs , 14. Actve costs . When apve costs , 23, 24 ve costs  other thve costs , 23, immve costs , 18, lowve costs , 16, no ve costs , low posve costs , likely ve costs . Modifieve costs ; those wve costs . The useve costs . The reave costs . Posterive costs , 15 withve costs . Within ve costs , 23, 24.ve costs . The secve costs , i.e. imve costs , 28, 29.ve costs , 28, 29.The current study supports laser frenotomy as an effective and safe treatment for newborns with ankyloglossia. Laser frenotomy caused low pain and very few complications both intra- and postoperatively with all wounds healed within thirty days with no recurrence of ankyloglossia. A significant improvement in breastfeeding and growth in newborns with a reduction of maternal nipple pain has also been shown.


Crimean-Congo hemorrhagic fever is a tick-borne and zoonotic emerging viral disease that is characterized by the sudden development of high fever and vascular bleeding in humans. The seroevidence among livestock has been reported, but it is less known in wild animals. Due to the importance and emerging state of the disease, we conducted a serosurvey on both domestic and wild animals in different areas of Turkiye. Serological investigations conducted on cattle, goats, and sheep revealed 10.81%, 15.15%, and 19.23% seropositivity, respectively, in the collected serum samples. We also found seropositivity rates in hare (23.81%) and wild boars (2.5%) indicating the substantial role of wild animals in virus epidemiology in Turkiye. This study provides first seroevidence of Crimean Congo hemorrhagic fever in wild animals in Turkiye. Hyalomma marginatum spp. has been identified as the natural reservoir and vector of the virus in Turkiye. A few studies have been conducted on domesticated animals showing the seroprevalence of CCHFV in them, but seroevidence in wild animals is absent. For contributing this antrum to the understanding of virus transmission in Turkiye, we performed a seroprevalence investigation of CCHFV in both wild and domesticated animals in various geographical areas of Turkiye. In-house IgG iELISA was performed for the screening of sera IgG in a total of 582 animal samples collected from boar (n = 40), cattle (n = 259), goat (n = 132), hare (n = 21), and sheep (n = 130). Results from ELISA performed on domestic animals revealed 10.81%, 15.15%, and 19.23% anti-CCHF virus seropositivity in cattle, goats, and sheep, respectively, in collected serum samples. ELISA tests performed in wild animals showed 23.81% and 2.5% positivity in hare and wild boars, respectively, suggesting the importance of wild animals in CCHF virus epidemiology in Turkiye. This study performed the first serological investigation of CCHFV in wild animals and provided the first seroevidence of CCHFV in wild boars and hare in Turkiye.Crimean-Congo hemorrhagic fever virus (CCHFV) is a zoonotic, tick-borne pathogen that is endemic to some parts of Europe, Africa, and Asia. The disease causes fever and hemorrhagic manifestations in humans but not in animals. Domestic and wild animals are asymptomatic hosts of CCHFV and are critical in the transmission cycle.  Hyalomma genus .All virus production and live virus handling took place in BSL-3 laboratories located at Firat University, Virology department . An in-house iELISA protocol was followed as described and optimized for IgG detection from different animal species ,25. The p = 0.367). However, seropositivity collected from sheep showed a significant difference (** p < 0.01) between Tokat and Yozgat cities, and this significance was higher in Yozgat (26.51%) than in Tokat (6.38%). Serum samples were obtained from various geographic areas, as presented in Seroepidemiological studies of CCHFV in animals provide evidence of the virus circulating in endemic regions and also help to identify the risk areas. Anti-CCHFV IgG has been detected in both domestic and wild animals in various endemic regions. Seroprevalence results without symptomatic infection from various animals show that animals can be infected with CCHFV. Serum IgG positivity in animals can last longer than asymptomatic viremia (7–15 days) ,21. SeroNairoviridae family. In fact, viral NP is the most abundant protein and is also known as an immune-dominant protein [Nairoviridae, there is a cross-reaction between Hazara virus NP and CHFV NP due to antigenic similarities [To measure seropositivity, a reverse passive hemagglutination-inhibition assay, virus neutralization assay, agar-gel diffusion precipitation, indirect immunofluorescence assay, and IgG ELISA were carried out for seroprevalence of CCHFV in animals . Due to  protein . This un protein . Marriot protein . In a relarities . Howeverlarities . Severallarities ,34. Nevelarities . Therefolarities . Here, wThe serological investigations were carried out on domestic animals from different countries, and the results were summarized by Spengler et al. . The nump < 0.01) when compared with Yozgat (26.51%). Such differences can be explained by ages of the animal sera collected, the foci of vector ticks, and the wild small animal population (hare), and even many other factors should be taken in consideration. For example, Tonbak et al. studied the distribution of tick species located in endemic foci-Kelkit Valley of CCHFV and identified that the most abundant tick species in domestic animals  was Rhipicephalus bursa followed by Hyalomma marginatum marginatum [Another seroprevalence study conducted in the Marmara district of Turkiye estimated 13%, 31.8%, and 66% seropositivity in serum samples collected from cattle, sheep, and goats, respectively, using serum neutralization assay . Sheep arginatum . Such diHyalomma m. marginatum tick species fed on infected hares (Lepus europaeus) and the virus was transmitted through transovarial and transstadial cycle in an experimental infection [Lapus spp.) [H. m. marginatum tick species in the field, and hence, human attack cases dropped from 100 to around 9 in the subsequent year. It was highlighted in the same review that adult ticks will most likely attack humans where inadequate large animals are prevalent [Although Turkiye has several wild animals in nature, no study has conducted a serological investigation of CCHFV in wild animals. In this study, we also evaluated the seroepidemiology of CCHFV in animals by collecting serum samples from hare and wild boars. If we consider smaller animals such as hares to serve as a host for immature ticks, it is not surprising that hares were found seropositive 23.81%) in this study. Early investigations by Zgurskaya et al. evidenced that nfection . In the nfection . Serologus spp.) . Seropos3.81% in us spp.) . Additious spp.) . On the us spp.) . These frevalent . Some strevalent ,43.Iberian ibex) showed higher seropositivity rates of 78.57% and 100% in Spain [Wild boars are considered large animals in the enzootic cycle of adult ticks. However, the number of serological studies is not satisfactory. This scarcity leaves a gap in knowledge regarding the role of wild boars in CCHFV epidemiology. Serological investigations from South Africa determined a 5% seropositivity ratio in warthogs . Neverthin Spain . The reaTo understand CCHFV epidemiology, wild animals and domestic animals need to be surveyed, since vector and reservoir ticks circulate the tick–host–tick lifecycle. Immature ticks feed preferentially on small animals , while adult ticks prefer larger mammals  . Based oIn this study, we provided the first seroprevalence results of CCHFV in wild animals, and we evidenced the serum IgG positivity in hares (23.81%) and wild boars (2.5%), which was comparable with domestic animals. We also screened domestic animals for IgG prevalence, and compared our results with those for wild animals  indicating that wild animals and livestock are equally important for circulating the CCHF virus in endemic areas such as seen in Turkiye.


Escherichia coli and T-like viruses under chemostat conditions, but the framework can be adapted to any microbe-virus system. Oligotrophic conditions led to smaller, lower-quality but more abundant hosts, and infections that were longer but produced a reduced viral offspring. Conversely, eutrophic conditions resulted in fewer but larger higher-quality hosts, and shorter but more productive infections. The virus influenced host evolution decreasing host size more noticeably for low than for high dilution rates, and for high than for low nutrient input concentration. For low dilution rates, the emergent infection time minimized host need/use, but higher dilution led to an opportunistic strategy that shortened the duration of infections. System collapses driven by evolution resulted from host failure to adapt quickly enough to the evolving virus. Our results contribute to understanding the eco-evolutionary dynamics of microbes and virus, and to improving the predictability of current models for host-virus interactions. The large quantitative and qualitative differences observed with respect to a classic description  highlights the importance of including viral plasticity in theories describing short- and long-term host-virus dynamics.Viruses play critical roles in the dynamics of microbial communities. Lytic viruses, for example, kill significant fractions of autotrophic and heterotrophic microbes daily. The dynamic interplay between viruses and microbes results from an overlap of physiological, ecological, and evolutionary responses: environmental changes trigger host physiological changes, affecting the ecological interactions of host and virus and, ultimately, the evolutionary pressures influencing the two populations. Recent theoretical work studied how the dependence of viral traits on host physiology  affects the evolutionarily stable host cell size and viral infection time emerging from coevolution. Here, we broaden the scope of the framework to consider any coevolutionary outcome, including potential evolutionary collapses of the system. We used the case study of  Viruses are the most abundant biological entities on Earth, yet cannot replicate without the synthesis machinery of a cellular host . This deEscherichia coli and T viruses using host growth rate as a measure of physiological state ).le trait , 33 has le trait :μmax(r)l ⋅ d−1) :k(r)=4πoverage) , we assuKN), therefore setting a tradeoff for the host. The parameter μmax, and μmax = 0 , the maturation rate, M (in virion ⋅ d−1), the burst size, B (in virion ⋅ cell−1), and the latent period, L (in d). Here, we considered the latter the focus of viral evolution, since it determines the timing of lysis and limits virion production by setting a maximum time for synthesis and assembly . Th. Thw lednditions . The sitw increased, as both latent period L and burst size B reached a lower value and thus the increase in dilution was not countered by an increase in viral production; instead, increased dilution led to a decrease in viral density that in turn enabled an increase in host density for high dilution rates. For the plastic case, the latent period also reached a lower value but the increasing host growth enabled a higher burst size; as a result, the viral population increased with dilution for mid-to-low input concentration N0, whereas the lower burst size B observed for high nutrient input led to a continuous decrease in viral availability. This lower viral density for high w and N0 does not result from the superinfection avoidance term, since similar behavior was observed for a version of the model without it . Host availability increased as dilution rate increased due to the improved growth conditions despite the improved viral performance, although higher input concentrations led to lower host densities as infection times shortened.As the dilution rate increased, host size, growth, and adsorption all increased and allowed for shorter infections, which led to a lower host density. Although the burst size decreased for the nonplastic case and slightly increased for the plastic case, in both cases the viral population also decreased, which points to host availability  driving this behavior. This decrease continued for the nonplastic case as In summary, both plastic and nonplastic cases showed a convex dependence of host density on dilution, with a minimum of host availability for intermediate dilution rates resulting from the balance between bottom-up and top-down pressures. This minimum was thus not plasticity-driven, although accounting for plasticity led to higher host and viral densities.rESS obtained in the absence of the virus as dilution increased means that, under low-growth conditions, the outcome of host evolution was regulated by both nutrient availability and viral pressure; however, under high-growth conditions, top-down regulation played no significant evolutionary role. Nonetheless, high input concentrations led to very large hosts in the absence of the virus, strategy that in the presence of the virus exposes the host to viral infection but increases host maximum growth rate under decreased competition for resources (i.e. higher top-down and reduced bottom-up pressures); in this case, host evolution was thus both bottom-up and top-down regulated for any dilution rate w. On the other hand, the convexity of the host density curve results from the eco-evolutionary dynamics with the virus, since the curve is instead concave in the absence of the virus (not shown).The convex trait curves comparing the outcome of coevolution with instances of single evolution obtained with this version of the model were in contrast with the concave curves obtained with the previous version . The facLnocoev, represents the latent period that “minimizes resources” for the virus, the departure from the 1:1 line indicates that other evolutionary strategies were prioritized by the virus under those environmental conditions. The dependence on dilution may stem from a shift from host availability dominating the viral evolutionary strategy when w is low, to host size and growth rate being the main influencing factor as w increases. This hypothesis is reinforced by the sensitivity of the curve to the input concentration N0 for high dilution rates observed only for the plastic case. For high nutrient input levels, the latent periods emerging for plastic and nonplastic cases agreed, as host growth remained around its maximum across dilution rates and thus any plastic viral trait remained effectively fixed. Another relevant factor to consider is that for high nutrient levels, and for the nonplastic case, demographic oscillations are more marked and thus the most suitable viral strategy may not be the one that prioritizes the minimization of resources but one accounting for the changing environment, which has been observed in microbes such as phytoplankton [For the virus, coevolution played an increasingly important role and shortened the emerging latent period as dilution rate and nutrient input increased. Given that the value in the absence of host coevolution, plankton .The modifications implemented in the new version of the model enabled more dynamical stability than previous iterations , 14, as L > 1 d) and the sequence of dominant phenotypes was approaching a lower L at the moment of host extinction. Given the proximity of the host size before extinction to the rESS reached without the virus, which we observed for many of these replicates, we speculate that extinction resulted from the host not being able to respond to changes in top-down regulation in a timely manner. The long infection times eased the evolutionary pressure on the host and, as a result, its evolving trait targeted the value expected under bottom-up regulation alone (i.e. virus not influencing host evolution). As host and virus evolved, infection time decreased, increasing mortality on the host, which could not adapt fast enough to the environmental changes and went extinct. This hypothesis is reinforced by the fact that increasing the dilution rate led to lower L values dominating at the moment of extinction: higher dilution means that the abiotic component of the environment exerts a higher pressure on the host and, therefore, lysis time needs to be smaller for the mortality rate due to lysis to become important for host dynamics. In the plastic case, the situation may be exacerbated by high growth rates from the dominant host guaranteeing high burst sizes for the virus. The evolutionary collapse is in this case a combination of an “evolutionarily underperforming” host and an “ecologically overachieving” (plastic) virus.In addition to these early extinctions, we also observed evolutionary collapses. In these cases, the host population collapsed first, followed by the viral population. Also common to these cases, the initial random viral phenotype showed a high latent period , or the directional flows present in the intestinal tract. The gut microbiome, for example, would be an example of high nutrient input and availability, and therefore our model predicts that the dominating sizes in the bacterial community in the absence of the virus would be much larger than in the presence of phage. This could be relevant for predicting the fate of a viral infection affecting the microbiome. A similar situation would apply to nutrient-rich parts of the oceans, like cold or coastal waters, or zones of upwelling. In oligotrophic environments, on the other hand, the expectation would be for microbial hosts to show smaller sizes than in the absence of viruses (e.g. lab cultures), prediction reinforced by the observed increased competitive ability of small cells when taking up nutrients , 57. OurThe evolutionary collapses we observed may provide useful information for the use of phages to eliminate bacterial infections (phage therapy) . Our resThese predictions could be tested with a comprehensive experiment that monitored the mid-to-long-term coevolution of host and virus, focusing on how host size and viral infection (offspring and duration) change over time and across environmental conditions. To the best of our knowledge, such an experiment does not exist. In addition to the obvious logistic challenges, one important factor complicating such an experiment is also a limitation of our framework: there are other potential targets for evolution in the system that may be subject to a higher pressure than host size and viral latent period. A situation that has been repeatedly studied in the past is the coevolutionary race that ensues when bacteria develop  immunity against viral infection, and the virus tries to overcome such immunity , 60, 61.The quantitative and qualitative differences observed here between the plastic and nonplastic versions of the model emphasize the need to consider plasticity in predictive theories for the eco-evolutionary dynamics of hosts and viruses. More empirical information is needed, however, to characterize viral plasticity in other important systems (e.g. different marine phytoplankton and virus species) and obtain the necessary expressions that unleash formalisms like the one presented here.S1 File(PDF)Click here for additional data file.


Our findings demonstrate that the TiO2-coated glass consistently inactivates coronaviruses upon contact under daylight illumination, in a time-dependent manner. A 99% drop in virus titer was achieved after 3.9 h. The electron micrographs of virus-covered TiO2-glass showed a reduced number of virions compared to control glass. Morphological alterations of TiO2-exposed viruses included deformation, disruption of the viral envelope, and virion ghosts, endorsing the application of this material in the construction of protective elements to mitigate the transmission of viruses. To the best of our knowledge, this is the first report showing direct visual evidence of human coronaviruses being damaged and morphologically altered following exposure to this photocatalyst.The antimicrobial properties of photocatalysts have long been studied. However, most of the available literature describes their antibacterial properties, while knowledge of their antiviral activity is rather scarce. Since the outset of the coronavirus disease 2019 (COVID-19) pandemic, an increasing body of research has suggested their antiviral potential and highlighted the need for further research in this area. In this study, we investigated the virucidal properties of a commercial TiOIMPORTANCE Surface contamination is an important contributor to SARS-CoV-2 spread. The use of personal protective elements and physical barriers  increases safety and has proven invaluable in preventing contagion. Redesigning these barriers so that the virus cannot remain infectious on them could make a difference in COVID-19 epidemiology. The introduction of additives with virucidal activity could potentiate the protective effects of these barriers to serve not only as physical containment but also as virus killers, reducing surface contamination after hand touch or aerosol deposition. We performed in-depth analysis of the kinetics of photocatalysis-triggered coronavirus inactivation on building glass coated with TiO2. This is the first report showing direct visual evidence (electron microscopy) of coronaviruses being morphologically damaged following exposure to this photocatalyst, demonstrating the high potential of this material to be incorporated into daily-life high-touch surfaces, giving them an added value in decelerating the virus spread. Since the outset of the coronavirus disease 2019 (COVID-19) pandemic, its etiological agent—severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)—has infected more than 445 million individuals worldwide and led to more than 5.9 million deaths , superoxide ions (O2·−) and hydrogen peroxide (H2O2). These reactive oxygen species (ROS) are hypothesized to exert their microbicidal action through oxidative stress, causing nucleic acid damage, lipid peroxidation, and biological membrane disruption , as well as measuring the kinetics of virus survival upon contact with this material.The antimicrobial properties of photocatalytic materials have been intensely studied. Some of the most prominent antimicrobial photocatalytic materials are TiOsruption . In thisCoronaviridae, genus Alphacoronavirus, subgenus Duvinacovirus). HCoV-229E virus has been widely used in coronavirus antiviral research. When exposed to different surface materials, it shows survival patterns similar to those of SARS-CoV and SARS-CoV-2 (Human coronavirus 229E (HCoV-229E) is a pathogen responsible for seasonal respiratory illness outbreaks .In this study, we sought to evaluate the virucidal activity of a TiO2-coated material was determined by 50% endpoint titration (50% tissue culture infective dose [TCID50] infectivity assays), where viruses exposed to TiO2 were compared to an equivalent virus inoculum exposed to bare (control) float glass for an equivalent interval.The virucidal assay consisted of inoculating a fixed amount of virus on the glass in the form of evenly distributed small droplets, as shown in Fig. S1A (lower left), and further covering the virus inoculum with a high-transmittance polyvinyl chloride (PVC) film . Each glass assembly was further incubated at room temperature for a specific interval according to the study time course. The infectious titer reduction  due to virus contact with the TiO50 assays by taking advantage of a recombinant green fluorescent protein (GFP)-expressing human coronavirus 229E strain. Since the host cells do not harbor the GFP coding sequence, any single green-fluorescent cell in the monolayer is considered virus infected, and the corresponding well in the plate is marked as positive during the titer calculation . While the absence of visually detected CPE  does not necessarily imply the lack of infection , the lack of green-fluorescent cells unambiguously indicates the absence of GFP expression and, thus, the absence of virus infection .Most infectivity assays rely on the ability of viruses to morphologically alter the infected cell monolayer (cytopathic effect [CPE]). These alterations may include but might not be limited to cell rounding and detachment, loss of monolayer integrity (confluence), appearance of cytosolic and/or nuclear inclusion bodies, refractivity, etc. . However, such alterations are often ambiguous, hard to identify, or difficult to distinguish from other morphological changes unrelated to infection, such as normal cell senescence, rendering this assay highly subjective. In our work, we overcame this limitation of conventional TCIDThree different illumination conditions were separately assayed: the virucidal experiment was performed on test surfaces in the absence of light, in the presence of D65 following preactivation with UV-A light, and in the presence of D65 without previous activation with UV-A light.2 in the commercial coated glass. The virus infectious titers recovered from both the coated and the control uncoated glass were similar during the time course of the experiment , except for the last time point, 48 h (P = 0.005)  were 11.1 h and 16.0 h for the commercial TiO2-coated glass and control float glass, respectively (t90%]). Further incubations for 73.8 h and 106.4 h are theoretically required to achieve 99% virus inactivation on both glasses, respectively (t99%)  . The incectively . Incubaty (t99%) .t99% from 48 to 96 h   , bars, fa et al. .2-coated glass when this surface was preactivated and further maintained under daylight (D65) illumination throughout the virucidal assay. The virus decay on control float glass under these conditions was of note and consistent with that observed during the virucidal experiment in the absence of light, with a half-life of 8.78 h and a t90% and a t99% of 29.2 h and 58.3 h, respectively  and continued to increase up to 26 h incubation . The virucidal activity attributed to the coating itself was calculated for each incubation time as the difference between the log titer of virus recovered from the control glass and that of the coated glass from the same time point. As the titers from the coated glass prominently dropped with incubation time, the virucidal activity indexes of these samples increased concomitantly, with statistically significant differences appearing at 6 h incubation  (As shown in < 0.001) , bars.2 coating against HCoV-229E/GFP fitted a biphasic kinetic model, with a well-defined exponential phase from the 2-h to the 8-h incubation time point and a stationary phase beyond 8 h, where no further significant decrease of virus titer was achieved , as can be seen in the expanded elapsed-time graph shown in t′99%) corresponding to 7.6 h , such as exposure to any surface over time.Interestingly, the virucidal activity of TiOto 7.6 h . This va2-coated glass against human coronavirus 229E/GFP are consistent with this kind of kinetic behavior, as discussed above  surfaces such as glass, stainless steel, and plastic . The auted above . Whether2 that supposedly limit its practical application. For instance, the relatively low band gap of its anatase phase (3.2 eV) implies that it can be photoactivated only with light with a wavelength of <387.5 nm (approximately 3% of the solar light spectrum) , i.e., 6, 7, 8, and 9 h incubation. The virucidal indexes ranged from 0.25 to 2.83 log virus titer reduction  . For thieduction . These veduction , suggestsviruses , hepatitsviruses  and inflsviruses . A possireported . Whether2 coating has an effect on the recovery of its genomic RNA after virucidal assays, the viruses washed off from both the coated and the control glass  were subjected to RNA extraction and quantification by reverse transcription-quantitative PCR (RT-qPCR). As shown in P > 0.05, two-way ANOVA or Kolmogorov-Smirnov nonparametric test), and they were also similar among control glass- and TiO2 glass-exposed viruses for all assay conditions and incubation time . The virus RNA quantity ranged from 8.31 × 1010 to 1.13 × 1012 copies per mL. This result was expected, because the loss of the integrity of viral envelope that ultimately leads to a drop in infectious titer has little or no effect on the amount of total viral RNA present in the sample, which can easily be detected via a sensitive technique such as RT-qPCR. In other words, the amount of virus genomic RNA in treated samples is not a good predictor of virucidal activity, as viral RNA could have been released from disrupted virions. In fact, the RNA might even be heavily damaged due to TiO2-induced oxidative stress and still be detected, as the RT-qPCR used in this study specifically targets a very short sequence within the virus genome. Our findings agree with previous data on SARS-CoV-2 RNA detection upon incubation with different materials  analyses of human coronavirus 229E/GFP incubated on float glass showed numerous virions of around 70 to 100 nm, when observed at a magnification of ×50,000 , left. Aaviridae .2-coated glass and further illumination with D65 light for 20 h led to a decrease in the number of total viral particles that were observed under SEM  increased between 2 and 26 h incubation for the TiO2-exposed viruses (Transmission electron microscopy (TEM) images of viruses recovered from 2- and 26-h incubation time points   showed a viruses .2 coating confers additional hydrophilicity to the glass upon chemical transformation during daylight irradiation. TiO2 coatings are capable of trapping water molecules after being irradiated with visible light in a time-dependent manner, thus becoming increasingly hydrophilic  of a particular material and its inactivating capacity against enveloped viruses. For example, the virus persists longer on hydrophobic materials such as polypropylene than on stainless steel (moderately hydrophilic), and it is also more stable on stainless steel and plastic than on glass and cotton/cellulose-based materials, which are considerably hydrophilic . Furtherrophilic , which cectivity .2-coated glass are one example of a material application with a potential epidemiologic impact. To the best of our knowledge, this is the first report showing direct visual evidence of human coronaviruses being damaged and morphologically altered following exposure to this photocatalyst, under scanning and transmission electron microscopes. Compared to regular disinfection agents, such as chlorine, alcohol, etc., the TiO2-based antiviral approaches have several advantages. First, the lack of toxicity of TiO2 makes it suitable for use in a wide range of environments and applications without compromising human health; second, as a catalyst, the material is not consumed in the reaction itself, which allows the coatings to function indefinitely. Where daylight-like illumination is present, such photocatalytic coatings could be incorporated onto daily-life high-touch surfaces, such as screens and urban furniture (furniture in public settings), thus giving them an added value not only in decelerating spread of the current pandemic but also in preventing future outbreaks.One of the best approaches to control the COVID-19 pandemic is to reduce virus transmission. Surface contamination has been acknowledged as an important contributor to SARS-CoV-2 spread. Therefore, in addition to antiviral drugs and vaccines, the use of personal protective elements and physical containment barriers  helps increase safety within closed environments and have proven invaluable in preventing contagion. Redesigning or engineering these barriers so that the virus cannot remain infectious could make a difference in SARS-CoV-2 and related pathogen epidemiology. The introduction of additives or coatings with virucidal activity could potentiate the protective effects of these safety elements to serve not only as physical barriers but also as virus “killers” to work upon contaminated hand touch- or aerosol-mediated surfaces. The virucidal properties found for this commercial TiOA recombinant human coronavirus 229E expressing the green fluorescent protein (HCoV-229E/GFP), constructed in the laboratory of Volker Thiel  , a gift 2 atmosphere incubator, for 60 to 72 h. After this incubation period, the free and cell-associated viruses were harvested together by scraping the monolayers into the culture medium. The preparations were frozen and thawed three times and clarified by centrifugation at 3,000 × g for 15 min in a Beckman Coulter Allegra X-22R centrifuge. The resulting supernatants were filtered through 0.45-μm polyvinylidene difluoride (PVDF) membranes and further centrifuged through a 30% sucrose 4-mL cushion, at 25,000 rpm in a Kontron Centrikon T-1180 ultracentrifuge (TST-28.38 rotor) for 2.5 h (4°C). Each virus pellet produced from two T150 flasks was resuspended in 200 μL Dulbecco modified phosphate-buffered saline (d-PBS).HCoV-229E/GFP high-titer virus stocks were produced by ultracentrifugation through a sucrose cushion as previously described for other enveloped viruses , with mi50)/mL. For the TCID50 assays, the appearance of at least one green-fluorescent (GFP-expressing) cell using the Eclipse Ts2-FL fluorescence microscope (Nikon) was considered indicative of a positive (infected) well, rather than the sole observation of virus-associated cytopathic effect. This fluorescence-based method allowed a more objective and accurate virus quantitation.Virus stock titrations were performed by the endpoint dilution method described by Reed and Muench  and the 2-coated glass  and bare glass  (5 cm by 5 cm by 0.35 cm) were obtained from Saint-Gobain Building Glass and were preactivated by UV-A light exposure for 4 h, using a QUV accelerated weathering tester (Q-lab). After preactivation, each glass specimen was placed inside a sterile 10-cm-diameter petri dish. The virucidal experiments were carried out according to the general guidelines of ISO standard 21702:2019 (6 TCID50 of HCoV-229E/GFP stock was deposited dropwise on each test surface and covered with a 4- by 4-cm piece of polyvinyl chloride (PVC) transparent film. The resulting assemblies were incubated at room temperature for 2, 4, 6, 7, 8, 9, 12, or 26 h, under illumination conditions that simulate daylight (D65). D65 illumination was generated with the aid of a CMlite model GLE-M5/17 lamp , placed inside the biosafety class IIB airflow cabinet. The light source was 25 cm away, above the glass (estimated maximum irradiance = 0.18 mW/cm2). After the indicated incubation times, the PVC film was separated with sterile tweezers, and the viruses from both surfaces were recovered by thorough washing with DMEM supplemented with 2% fetal bovine serum . A similar procedure was done in the presence of D65 light in which the glass specimens were not subjected to UV-A preactivation. A control experiment in the absence of any type of light was also performed.TiO702:2019 , with mi2), the monolayers were observed under the fluorescence microscope, and the number of wells per dilution showing at least one green-fluorescent (infected) cell was recorded. The dilution infecting 50% of wells (TCID50) was calculated as described by Reed and Muench . A virucidal index for each time point was calculated by subtracting the logarithm of virus titer found in the test glass from the logarithm of virus titer recovered from the paired control glass.The residual infectivity of recovered viral particles was determined using the endpoint titration method. For this purpose, serial 10-fold dilutions from the samples were made and used to inoculate HuH-7 confluent monolayers in 96-well plates, with 100 μL/well using 6 wells per dilution (sextuplicate). After 72 h incubation , 5′-GTCAGCAATGGAAAGACCCTTAGT-3′ (reverse primer), and 5′-TAGCAAACTTGTTACTTCTGGACTTGGCACTGTG-3′ (TaqMan probe labeled with 5′ ABY dye and the 3′ nonfluorescent proprietary quencher QSY) (Thermo Fisher Scientific). The primers and probe were based on the human coronavirus 229E reference sequence in GenBank (accession number NC_002645) and were used at 450 nM and 125 nM, respectively. The ROX (carboxyrhodamine) dye present in the master mix was used as the passive reference, and the cycling conditions were those recommended by the TaqMan master mix’s manufacturer. For absolute (copy number) quantification of samples, a standard curve was generated  by amplifying (in triplicate) serial decimal dilutions of HCoV-229E/GFP RNA isolated from purified virions and quantifying with the aid of a Qubit 2.0 fluorometer (Invitrogen).The RNA from viruses recovered after time course incubations on both test surfaces  was extracted with the RNeasy minikit (Qiagen) and quantified by RT-qPCR using the TaqMan Virus Fast one-step master mix (Applied Biosystems) in a QuantStudio 5 real-time PCR thermocycler (Applied Biosystems). For this purpose, the following oligonucleotides were designed with the aid of Primer Express 3.0.1 software (Applied Biosystems): 5′-4 TCID50 of HCoV-229E/GFP were evenly deposited on each surface (TiO2-coated glass and bare float glass) and incubated in the presence of D65 light for 20 h. Afterwards, both surfaces were sputter coated with gold by using a Balzer SCD004 sputter coater and examined through a JEOL model JSM-6610LV scanning electron microscope, with a 20-kV acceleration voltage and a working distance of 10 mm.For scanning electron microscopy (SEM) analyses, 5 × 102-coated glass, after the virucidal experiment , were deposited on a petri dish. A Formvar-copper 200 mesh grid  was floated on each sample drop, Formvar side down, for 10 min. Afterwards, the grids were blotted on their sides on filter paper to remove the liquid excess and were further floated  for 1 min on a 4% uranyl acetate solution. The grids were blotted again, allowed to dry overnight, and imaged using a JEOL model JEM-1011 electron microscope. Samples were micrographed at ×100,000 and ×150,000 magnifications, with an acceleration voltage of 100 kV.The biological specimens for transmission electron microscopy (TEM) were prepared as follows: 20-μL drops of virus recovered from control and TiOt50%) and time to reduce titers by 1 log unit (t90%) and 2 log units (t99%) were calculated by using regression analyses from the time course curves generated from data. The experiments were performed in triplicate, and virus titers (log TCID50/mL), virucidal indexes, and RNA copy numbers collected across time points were assessed for normality and homoscedasticity using the Shapiro-Wilk and the Levene tests, respectively. P values of >0.05 were considered indicative of data normality and variance homogeneity. Multiple mean comparison analyses were performed with ANOVA. Paired-means comparisons were performed using Student's t test, unless otherwise indicated. P values of <0.05 were considered indicative of significant differences among means. The different means were assessed using Sidak’s or Tukey’s post hoc multiple-comparison tests. All experiments were performed in triplicate, and data are presented as means and standard deviations. All statistical analyses were done using GraphPad Prism v7 software.For each glass material, parameters such as intrinsic virus half-life (The data sets generated in this study can be found in the paper.