Botrytis cinerea. Transmission electron microscopy of infectious hyphae and hyphae grown in vitro revealed extracellular vesicles of various sizes and densities. Electron tomography showed the co-existence of ovoid and tubular vesicles and pointed to their release via the fusion of multi-vesicular bodies with the cell plasma membrane. The isolation of these vesicles and exploration of their protein content using mass spectrometry led to the identification of soluble and membrane proteins involved in transport, metabolism, cell wall synthesis and remodeling, proteostasis, oxidoreduction and traffic. Confocal microscopy highlighted the capacity of fluorescently labeled vesicles to target cells of B. cinerea, cells of the fungus Fusarium graminearum, and onion epidermal cells but not yeast cells. In addition, a specific positive effect of these vesicles on the growth of B. cinerea was quantified. Altogether, this study broadens our view on the secretion capacity of B. cinerea and its cell-to-cell communication.Fungal secretomes are known to contain a multitude of components involved in nutrition, cell growth or biotic interactions. Recently, extra-cellular vesicles have been identified in a few fungal species. Here, we used a multidisciplinary approach to identify and characterize extracellular vesicles produced by the plant necrotroph  Filamentous fungi are the most prevalent plant pathogens, causing dramatic reductions in the yield and quality of agricultural and horticultural production annually . During EVs are released by bacterial, fungal, plant and animal cells ,6,7. TheEVs constitute a heterogeneous population of vesicles that can be sorted in three classes: exosomes 30–150 nm), micro-vesicles/ectosomes (100–1000 nm) and apoptotic micro-vesicles (800–1000 µm) or bodies (>1 µm) ,15,16,170 nm, micSaccharomyces cerevisiae and the yeast human pathogen Cryptococcus neoformans also suggest that exophagy (fusion of autophagosome with the plasma membrane) and components of the conventional and non-conventional secretory pathways of cells are also involved [The biogenesis of EVs is still unclear at the molecular level. It has been proposed that micro-vesicles shed from the plasma membrane or originate from inverted macropinocytosis, while exosomes originate as intra-luminal vesicles inside the endosome (during the maturation of the latter into multi-vesicular bodies (MVB)) and are released upon fusion of the MVB with the plasma membrane ,20,21,22involved ,30,31,32Aspergillus nidulans [C. neoformans [C. neoformans in 2007 [Alternaria infectoria, a ubiquitous environmental fungus that both spoils food crops and causes human infections [In fungi, EVs were first observed in the early 1970s using transmission electron microscopy (TEM) at the surface of protoplasts of the fungus nidulans  and at toformans . In paraoformans , and latoformans . Togetheoformans , these o in 2007 . EVs werfections . They hafections ,41,42,43S. cerevisiae [Fusarium oxysporum and F. graminearum, carrying proteins and effectors involved in virulence [Colletotrichum higginsianum, the causal agent of anthracnose disease [Mostly investigated in human pathogenic species and in revisiae ,50,51,52revisiae ,43,53,54revisiae ,50, pigmrevisiae ,43 and arevisiae . In the revisiae ,56,57,58irulence ,60. EVs  disease .B. cinerea is an ascomycetous fungus responsible for gray mold disease. It can infect hundreds of dicotyledonous plants and was listed among the ten most devastating plant pathogenic fungi worldwide [B. cinerea is important to understanding its pathogeny. This study explores the production of EVs by B. cinerea and their potential roles in its biology.orldwide . The necorldwide  that kilB. cinerea (teleomorph Botryotinia fuckeliana (de Bary) Whetzel) was maintained on sporulation medium (MS) [F. graminearum was maintained in 25% V8 vegetable juice, 3.75 g/L CaCO3, and 25 g/L agar. For TEM ultrastructure of B. cinerea grown in solid culture: mycelium explants were used to inoculate MS plates overlaid with cellophane sheets and incubated for 3 days at 21 °C in the dark. The cellophane was then transferred to a clean medium-free Petri dish for fixation. For TEM ultrastructure of B. cinerea grown in liquid culture: mycelium explants were used to inoculate ½ PDB or conidia (105/mL) were used to inoculate a minimum medium  glucose). Cultures were agitated for 4 days at 110 rpm, with medium renewal on day 3. The mycelia were filtered (100 µm) prior to fixation.All strains were grown at 21 °C. Strain B05.10 of ium (MS)  and grow5 conidia per plate) the surface of cellophane sheets overlaying solid MS [For EVs production, conidia were collected from 11-day-old cultures on MS and used to inoculate  sorbitol, 1 mM EDTA and 10 mM Tris-HCl pH 7.4, and a discontinuous gradient (5–40%) was formed by layering 3 mL of each solution in polypropylene centrifugation tubes. The vesicles were loaded onto this gradient and centrifuged at 100,000 g for 16 h . Fractions (2 mL) were collected from the top of the gradient and their density was determined using spectrophotometry (244 nm) according to the manufacturer’s instructions . Protein concentrations were obtained using QubitTM 2 fluorimeter  and the QubitTM protein assay kit according to the manufacturer’s instructions, or with BCA protein assay  for CFSE-BcEVs. The fraction of interest (fraction 5) was washed twice in PBS , suspended in 300 µL of cold PBS, and stored on ice before use, or it was placed at −80 °C for conservation.All steps were performed at 4 °C. The mycelium was harvested and gently rotated for 4 h in 300 mL of cold PBS . Following filtration (200 µm), cell debris were removed from the filtrate by two successive centrifugations . The resulting supernatant was filtered twice (40 µm and 1.2 µm) and centrifuged for 1 h at 100,000 g . The pellet containing the vesicles was solubilized in 2 mL of cold PBS. OptiPrep™  60% The 100,000 g pellet was suspended in 2 mL of cold PBS. Carboxyfluorescein succinimidyl ester  was added (40 µM) and the sample was incubated for 1h in the dark at 21 °C before loading onto the OptiPrep™ gradient. The fluorescence in the recovered fractions was measured using a TECAN infinite M1000 . In parallel, PBS (2 mL) containing 40 µM CFSE  was subjected to the same protocol, and fraction 5 was recovered to be used as PBS control sample.Particle size distribution of EVs preparations was determined by dynamic light scattering (DLS) at 19 °C using a Zetasizer nano series . Eight biological replicates and three technical replicates (ten reads each) were performed.Sample Preparation—Mycelia were fixed in 4% paraformaldehyde (PFA) and 0.4% glutaraldehyde (GA) in 0.1 M PHEM  for 30 min at room temperature (RT) with gentle shaking. The buffer was discarded, and a second incubation  was performed in 0.1 M PHEM buffer containing 2% PFA and 0.2% GA. High-pressure freezing was performed as described in [4, the samples were slowly warmed to −60 °C (2 °C/h), stored for 10 h at −60 °C. Then, the temperature was raised to −30 °C (2 °C/h), the samples were stored for a further 10 h at −30 °C before a 1 h incubation at 0 °C, and storage at −30 °C until further processing . Then, the samples were rinsed 4 times in pure acetone before being infiltrated with progressively increasing concentrations of resin  in acetone while increasing the temperature to 20 °C. At the end, pure resin was added at RT and the samples were placed at 60 °C for 2 days of polymerization.ribed in . BrieflyTransmission Electron Microscopy (TEM) of fungal cells grown in vitro—70 nm thin sections were cut from high-pressure-frozen-resin-embedded samples, using a UC7 ultramicrotome  and collected on 100 mesh Formvar carbon copper grids. The sections were post-stained with 2% uranyl acetate and lead citrate (5 min each). Samples were observed using a Tecnai G2 Spirit BioTwin microscope  operating at 120 kV with an Orius SC1000B CCD camera .Electron tomography—For electron tomography, 200–350 nm thick sections were used. Tomogram tilt series were collected at ×6500 from −40° to +40° or ×9600 from −50° to +50° with 2° intervals on a Tecnai TF20 microscope operating at 200 kV  and using a CCD (4 k × 4 k) camera . Tomography data acquisition, automated centering, focus adjustment, tilt setting, and image capture were performed using the SerialEM software 3.8.0 beta [8.0 beta  while coTEM of fungal cells grown in planta—Infection assays were performed using primary French bean leaves (Phaseolus vulgaris var Saxa) inoculated with conidia (104/mL in ¼ PDB) and incubated 40 h at 21 °C under 80% relative humidity and dark–light (16 h/8 h) conditions. Infection zones were collected and fixed in 0.1 M phosphate buffer, pH 6.8, containing 2.5% GA and 4% PFA . Following 6 washes in 0.2 M phosphate buffer, pH 6.8 , 2 mm leaves band were cut out, treated with 2% osmium in 0.1 M phosphate buffer, pH 6.8 , rinsed in water (3× 30 min) and dehydrated through incubations in 30, 50, 70, 80, 90, 95% ethanol baths (2× 15 min each) and in 100% ethanol (3× 10 min). The samples were then impregnated through three successive Ethanol/Spurr’s resin solutions (ratio (v/v) 3/1 (2 h), 1/1 (2 h) and 1/3 (3 h)). The leave bands were heated to 70 °C for 3 days in Spurr’s resin for polymerization on silicon molds. Ultra-thin sections were cut and observed onto TEM–copper–Formvar grids (75 meshs) with a TEM Philips CM120 operating at 120 kV.TEM of isolated EVs—Five μL of EVs suspension in PBS were deposited onto a TEM–copper–Formvar grid and left decanted for 30 s. Excess liquid was absorbed using Whatman paper and the grid was flipped over a drop of 2% uranyl acetate (UA) for 30 s. UA was removed and the grid was washed with distilled water. The grid was then placed into the chamber of a transmission electron microscope JEOL 1400 Flash.Confocal microscopy—Observations were performed in 96-well micro-plates . Conidia of B. cinerea or F. graminearum were suspended in 200 µL (5 × 103/mL) of ¼ PDB and incubated at 21 °C in the dark, for 6 h, 10 h or 14 h. After removal of 160 µL of medium, 30 µL of CFSE-BcEVs (Fraction F5) in PBS buffer were added, and the plate was agitated (300 rpm) at 21 °C in the dark for 1–4 h. The control experiments used 30 µL of the PBS-CFSE (Fraction 5). When used, 20 µL of S. cerevisiae cells (2.5 × 105/mL), grown in ¼ PDB at 28 °C and collected during the exponential phase, were added. Aniline blue  was directly added to the samples from a 10× solution in water. For onion epidermis observations, 1.5 cm2 of epidermis was deposited onto 10 µL of CFSE-BcEVs (fraction F5) on a microscope slide and incubated in the dark for 3 h at 21 °C. Before observation, the onion epidermis was rinsed with PBS and mounted with a coverslip. All observations were performed with a Zeiss axio Observer 7 LSM-800 confocal microscope (Zen 2.6 Blue software) set at 493 nm (excitation) and 817 nm (emission) for CFSE detection and at 353/465 nm for aniline blue detection.B. cinerea or F. graminearum were suspended in 100 µL PDB (5 × 103/mL) in 96-well micro-plates, and incubated at 21 °C for 2 h in the dark. After the removal of 75 µL of medium, 50 µL of sterile water and 25 µL of BcEVs (Fraction 5) representing 5.5 ± 1.8 µg of total protein (BCA protein assay) were added. The plate was incubated at 21 °C and growth was measured over time at 620 nm using a TECAN infinite F200Pro.Conidia of m/z 375–1500) were recorded at a resolution of R = 120,000 (@ m/z 200) and an AGC target of 4 × 105 ions collected within 50 ms, followed by a top speed duty cycle of up to 3 s for MS/MS acquisition. Precursor ions (2 to 7 charge states) were isolated in the quadrupole with a mass window of 1.6 Th and fragmented with HCD@30% normalized collision energy. MS/MS data were acquired in the ion trap with rapid scan mode, AGC target of 3 × 103 ions and a maximum injection time of 35 ms. Selected precursors were excluded for 60 s. Protein identification and label-free quantification (LFQ) were carried out using Proteome Discoverer 2.4. MS Amanda 2.0, Sequest HT, and Mascot 2.5 algorithms were used for protein identification in batch mode by searching against a ENSEMBL B. cinerea ASL83294v1 database . Two missed enzyme cleavages were allowed for the trypsin digestion. Mass tolerances in MS and MS/MS were set to 10 ppm and 0.6 Da. Oxidation (M) and acetylation (K) were searched as dynamic modifications and carbamidomethylation © as a static modification. Peptide validation was performed using Percolator algorithm [p-value < 0.05). The mass spectrometry proteomics data were deposited in the ProteomeXchange Consortium via the PRIDE [Extracellular vesicles preparations were concentrated to 40 µL on Vivaspin 500 , solubilized in Laemmli sample buffer  and boiled (100 °C) for 3 min. Samples with an equal protein concentration (10 µg) were loaded onto a 10% acrylamide SDS-PAGE and the migration was stopped when the proteins entered the resolving gel. The steps of sample preparation and in gel digestion by trypsin were performed as previously described . NanoLC-lgorithm  and onlyhe PRIDE  partner B. cinerea protein lists, including plant-cell-wall-degrading enzymes (PCWDE), fungal cell wall enzymes (FCWE), transporters, purine metabolism, virulence factors, proteases, laccases [Functional enrichment Gene Ontology (GO) analyses were performed using gProfiler ,71. Putalaccases ,78,79. Tlaccases .B. cinerea to produce EVs was first investigated using transmission electron microscopy (TEM). To this end, the fungus was grown in liquid medium using conidia or mycelial explants as an inoculum. In parallel, the fungus was also grown on solid medium overlaid with cellophane. In hyphae collected from all cultures, vesicles could be detected using TEM between the fungal plasma membrane and the cell wall. Groups of vesicles heterogeneous in size (50–500 nm) and in electron densities were detected , as shown by TEM. Unfortunately, a gelatinous material that co-sedimented with these particles hampered their manipulation too much for this protocol to be adopted.B. cinerea was then grown on cellophane sheets overlaying solid media and the secreted material was recovered via gentle agitation of the mycelium detached from the cellophane in PBS buffer. Filtration, differential centrifugations and additional separation through an iodixanol density gradient allowed one fraction out of six -containing proteins  highlighting localization, transport and beta-glucan biosynthesis  into eight functional categories (Predictions of transmembrane (TM) domains and glycosylphosphatidylinositol (GPI) anchors were used to list 271 proteins containing 1 to 16 putative TM and/or a GPI anchor, representing 40% of the 673 proteins of interest . Gene onynthesis A. Predicynthesis B. Based ynthesis C. Addititegories : transpotegories D. Sub-categories D.p-value < 7.2 ×Among the 673 proteins of interest, 402 (60%) were predicted not to contain any TM domain or GPI anchor. As shown in us (13%) B. The reus (13%) C. Finallus (13%) D. Five cus (13%) .B. cinerea [Only one known virulence factor (BcSun1) and one cell-death-inducing protein (BcGs1) were found in the 673 BcEV protein of interest. No candidate effector protein could be identified in this dataset when the Effector-P prediction tool was used. However, BcGs1 is described as an effector of  cinerea  and five cinerea   were deposited onto onion epidermis fragments. Onion epidermis is an established experimental model for evaluating plant penetration by croscopy A. A fluocroscopy B.B. cinerea was then explored. First, BcEVs were deposited onto bean leaves. In comparison to the mock experiment, this triggered no visible reaction of the plant. To circumvent a possible limitation of BcEVs penetration through the plant cuticle, infiltration assays on tobacco leaves were then performed. The leaves showed no response to BcEVs, whereas the yellowing of their tissues was visible when the raw B. cinerea secretome was infiltrated as a positive control  collected in exponential phase were added together with CFSE-BcEVs to the germinated conidia of B. cinerea, the periphery of the B. cinerea cells was stained within an hour, but that of the yeast cells was not , BcEVs of diverse shapes, sizes and electron densities can be observed between the cell wall and the plasma membrane of hyphae grown in liquid or solid media. Electron tomography further revealed that BcEVs co-exist as discrete vesicles and nanotubes, structures not yet reported in pathogenic fungi but observed in humans  and in soformans , includioformans .C. neoformans is grown in liquid medium [C. neoformans [BcEVs collected by differential centrifugations of liquid culture supernatants formed gelatinous pellets that were very difficult to manipulate. Considering the EVs contamination by glucurono-xylomannan when d medium , it is pd medium . It yieloformans , the prehttp://exve.icc.fiocruz.br, accessed on 15 December 2021). This is an intermediate situation between the 3312 and 3037 proteins found in C. neoformans and in A. fumigatus on the one hand, and the 1410 and 1256 proteins found in Histoplasma capsulatum and in C. albicans on the other hand. Based on the low variation in abundance between 4 biological replicates, 673 of these proteins were selected as the best candidates of BcEV proteins. The predicted distribution and functions of these proteins are very similar to those of the proteins identified in EVs produced by mammalian cells and fungi [C. albicans [A total of 2461 proteins were identified in BcEVs. According to the Fungal EV database 2021 , as well as cell-wall-related proteins or the sugars, lipids/sterol, amino-acids and phosphate-related metabolic enzymes could stimulate the growth of B. cinerea cells. The same applies to the ribosomal proteins, to proteins involved in traffic or proteostasis, or to enzymes of the respiratory chain. However, the reason why these proteins would not operate in F. graminearum cells is unclear.The impact of BcEVs on recipient cells can derive from either, or both, surface and lumenal compounds. For instance, the membrane chitin and glucan synthases can rescue yeast cells from antifungal molecules , while pB. cinerea. This multidisciplinary approach revealed complementary biological, biochemical and functional information. These EVs are heterogeneous in shapes, sizes and electron densities. Similar to EVs from human fungal pathogens, they are carriers of both membrane and soluble intracellular and extracellular proteins involved in diverse biological processes. Their potential role in intra and interspecies communication has been revealed. How this communication affects the recipient cells remains to be elucidated.In conclusion, this study documents the production and isolation of EVs by


Athelia rolfsii, which cause root rot disease, were investigated. The findings revealed that Zn and B application to the potting soil alleviated the adverse effect of A. rolfsii on sugar beet plants and increased the chlorophyll content in leaves. The increased enzymatic antioxidant activities such as catalase (CAT), peroxidase (POX), and ascorbate peroxidase (APX), and non-enzymatic antioxidants such as ascorbic acid (AsA) were observed in Zn applied plants compared to both uninoculated and inoculated control plants. A significant rise in CAT activity was noted in both leaves (335.1%) and roots (264.82%) due to the Zn2B1.5 + Ar treatment, in comparison to the inoculated control plants. On the other hand, B did not enhance the activity of any one of them except AsA. Meanwhile, A. rolfsii infection led to the increased accumulation of MDA content both in the leaves and roots of sugar beet plants. Interestingly, reduced MDA content was recorded in leaves and roots treated with both Zn and B. The results of this study demonstrate that both Zn and B played a vital role in A. rofsii tolerance in sugar beet, while Zn enhances antioxidant enzyme activities, B appeared to have a less pronounced effect on modulating the antioxidant system to alleviate the adverse effect of A. rolfsii.Generation of reactive oxygen species (ROS) constitutes an initial defense approach in plants during pathogen infection. Here, the effects of the two micronutrients, namely, zinc (Zn) and boron (B), on enzymatic and non-enzymatic antioxidant properties, as well as malondialdehyde (MDA) contents in leaves and roots challenged with  Beta vulgaris L.) holds significant global importance as a cultivated sugar crop, ranking second only to sugarcane [Athelia rolfsii (Curzi) Tu and Kimbrough . This fungus primarily infects the crown area or regions near the stem close to the soil surface, resulting in the decay of infected tissues. Such infections can lead to substantial yield losses, often reaching up to 50% [A. rolfsii significantly disrupts proteins and metabolites associated with both photosynthesis and antioxidant mechanisms within chloroplasts and mitochondria, impacting carbohydrate and amino acid metabolism processes [Sugar beet , superoxide (O2•−), hydrogen peroxide (H2O2), and hydroxyl radical (OH•) is one of the defense mechanisms employed by plants against pathogens. However, an excess of ROS inside the plant cell can lead to cellular damage. To mitigate this risk, plant cells generate various types of antioxidants to reduce the level of ROS. This detoxification involves both enzymatic and non-enzymatic antioxidant constituents [Plants activate many defense mechanisms in reaction to biotic stress, as it may be linked either directly to responses to the immune system or to a signal transduction pathway. Production of ROS such as singlet oxygen  content in cells, which is a secondary product produced during lipid peroxidation [Superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), glutathione peroxidase (GPX), and peroxidase (POX) are enzymatic antioxidants, while ascorbic acid (AsA) and glutathione (GSH) are non-enzymatic antioxidants. These enzymes are present in nearly all subcellular units. Generally, an organelle contains numerous enzymes capable of scavenging ROS . The asc cytosol . Similarxidation ,16.Zinc and boron are two micronutrients that have been reported to be essential for normal plant growth and for protecting plants against adverse environments . Of themA. rolfsii, exhibiting root rot symptoms. Notably, the application of Zn and B in sugar beet plots partially suppressed root and crown rot symptoms in the sugar beet field (unpublished data). Despite sclerotium root rot posing a significant challenge in sugar beet cultivation worldwide, the potential for disease control through the utilization of Zn and B has not previously been investigated. A recent study conducted by Rerhou et al. [A. rolfsii in sugar beet plants remain undisclosed. It is conceivable that these micronutrients might trigger the activation of the antioxidant system within sugar beet plants, assisting the plants in fortifying their defense mechanisms against A. rolfsii. Consequently, investigating the roles of Zn and B could present a promising avenue to diminish the incidence of root rot disease caused by A. rolfsii, thereby bolstering resistance, and promoting plant growth. The present study evaluated the contributions of Zn and B to the activation of the defense mechanisms in sugar beet plants against A. rolfsii.Recently, Paul et al.  assessedu et al.  has shed2) were used to conduct the study. The study consisted of the following treatments- (i) uninoculated control (Zn2B0), (ii) inoculated control , (iii) Zn2B0 (Zn 2.0 kg ha−1), (iv) Zn2B0 + Ar, (v) Zn4B0 (Zn 4.0 kg ha−1), (vi) Zn4B0 + Ar, (vii) Zn0B1.5 (B 1.5 kg ha−1), (viii) Zn0B1.5 + Ar, (ix) Zn0B3 (B 3 kg ha−1), (x) Zn0B3 + Ar, (xi) Zn2B1.5, (xii) Zn2B1.5 + Ar, (xiii) Zn2B3, (xiv) Zn2B3 + Ar, (xv) Zn4B1.5, (xvi)Z4B1.5 + Ar, (xvii) Z4B3, and (xviii) Z4B3 + Ar.The experiment was conducted at the Agronomy Field Laboratory, Department of Agronomy, Bangladesh Agricultural University, Mymensingh, from November, 2019 to February, 2020. The tropical sugar beet variety PAC-60008 was used for the experiment. The study was laid out in a randomized complete block design (RCBD) and replicated three times. Similar size of plastic pots  0.4 dS/m, organic carbon 1.00%, N 0.09%, P 1.60 ppm, K 0.10% meq/100g soil, Ca 8.30 meq/100 g soil, Mg 3.29 meq/100 g soil, S 2.98 ppm, Zn 0.21 ppm, and B 0.23 ppm. The soil was properly air dried, crushed with a wooden hammer, and thoroughly mixed. It was then cleaned and autoclaved. Finally, an equal amount of soil (10 kg) was used to fill each pot. Inorganic fertilizers were mixed thoroughly with the soil during pot filling . Seeds wA. rolfsii isolate BTSB2 was used for inoculation assay [A. rolfsii culture were cut and placed into the flask containing wheat seeds. The flask was then incubated for 10–15 days at 25 °C with a 12-h photoperiod. Infected wheat seeds were placed adjacent to the collar region of sugar beet plants 42 days after emergence and leaf and root samples were collected for analysis 7 days after the fungal inoculum application. The survival rate of the inoculated plants was calculated 20 days after the pathogen application and expressed as a percentage.The on assay . Fungal on assay . BrieflyA transportable SPAD meter  was used to determine the greenness (chlorophyll content) of sugar beet leaves 7 days after pathogen inoculation. Five fully expanded new leaves were selected SPAD values recording of beet plants. The average data were collected from the middle portion of the five leaves per plant between 9:00 to 10.00 a.m. [2PO4 buffer  for POX and APX analysis. The homogenized samples were collected and centrifuged at 5000 rpm for 20 min at 4 °C. Assay of antioxidant enzymes  was conducted using the supernatant.Two plants from each replication  were taken and separated into shoots and roots. The collected leaf and root samples were then used for further analysis. Initially, 1 g of green leaves and roots was thoroughly blended and homogenized in a pre-chilled mortar-pestle, adding 5 mL of Tris–HCl buffer  for CAT and KH2O2 (20 mM), and 100 μL of enzyme extract (supernatant). The H2O2 was added to start the reaction and the CAT activity was measured at 240 nm wavelength.The CAT activity was determined following the technique described by Aebi . The rea2PO4 buffer , EDTA (0.1 mM), H2O2 (0.1 mM) and guaiacol (10 mM). The reaction was initiated by adding 50 µL of sample solution to the reaction buffer (950 µL). The activity was determined at a wavelength of 470 nm for 30 sec, using an extinction coefficient 26.6 mM−1 cm−1.Activity of POX was measured according to the method described by Nakano and Asada . The rea2PO4 buffer , EDTA (0.1 mM), H2O2 (0.1 mM), and 0.5 mM ascorbate to the enzyme extract (50 µL). The activity was measured at 290 nm wavelength for 1 min, using an extinction coefficient 2.8 mM−1 cm−1 [For the determination of APX activity, the reaction mixture was prepared by adding KHM−1 cm−1 .The AsA content was calculated following the method described by Rangana . The pla−1 cm−1 was applied to calculate the MDA, expressed in µmol g−1 fresh weight [Oxidative damage in the leaf and root cells was estimated as the content of total 2-thiobarbituric acid (TBA)-responsive substances and expressed as equivalents of MDA following the method of Cakmak and Horst . The amoh weight .The statistical analyses were performed using the software package MSTAT-C, and mean differences were assessed using Duncan’s Multiple Range Test (DMRT) .0B0), although some yellowing of leaves was observed. On the other hand, A. rolfsii (inoculated control) treated plants showed wilting within 6–10 days after inoculation. Plants died within 20–25 days after inoculation, and infected roots became rotten and covered with mycelial mass  recorded in the inoculated control compared to the uninoculated control (25.03). Elevated chlorophyll content was recorded in plants treated with Zn4B1.5 (69.50) which was 177.67 and 266.56% higher in comparison to the uninoculated and inoculated control, respectively . Interestingly, decreased CAT activity was detected in uninoculated B applied plants but increased in pathogen inoculated plants. The lowest CAT activity was recorded in plants treated only with pathogen (Zn0B0 + Ar), measuring 45.06 µmol min−1 g−1 FW. The highest CAT activity (196.34 µmol min−1 g−1 FW) was recorded for Zn2B1.5 + Ar which was 335.1% higher than inoculated control (The CAT activity in leaves and roots was significantly influenced by various treatments A. In the control A.−1 g−1 FW) was noticed in the roots of Zn4B0 + Ar treated plants, which was 264.82% higher than the inoculated control plants, followed by Zn2B0 + Ar (68 µmol min−1 g−1 FW), Zn2B1.5 + Ar (61.75 µmol min−1 g−1 FW), Zn2B3 + Ar (58.84 µmol min−1 g−1 FW) and Zn4B3 + Ar (58.09 µmol min−1 g−1 FW) treated plants. Boron treatments did not enhance CAT activity in sugar beet roots. CAT exhibited a substantial decline in its activity in inoculated control (Zn0B0 + Ar) compared to the uninoculated plants  and inoculated (Zn0B0 + Ar) control, while decreased POX activity was detected in plants treated only B but it increased in pathogen inoculated plants. The highest POX activity (1.96 µmol min−1 g−1 FW) was observed in Zn2B1.5 + Ar treated plants, which was 43% higher than inoculated control (1.37 µmol min−1 g−1 FW) in leaves. The lowest POX activity was found in Zn0B3 (0.53 µmol min−1 g−1 FW) (The sugar beet plants exhibited a similar pattern of POX activity as CAT in response to the application of Zn and B under  g−1 FW) A.−1 g−1 FW and 12.23 µmol min−1 g−1 FW) was observed in Zn2B0 and Zn2B1.5 treated plants with pathogen inoculation, which was 26.05% and 17.14% higher than the inoculated control. The lowest POX activity was recorded in Zn0B1.5 (4.36 µmol min−1 g−1 FW) and Zn0B3 (3.81 µmol min−1 g−1 FW) treated plants  was recorded for Zn2B0 + Ar, which was 109.75% higher than inoculated control, whereas the lowest APX activity was recorded in Zn0B1.5 (0.37 µmol min−1 g−1 FW)  A.2B1.5 + Ar (13.27 µmol min−1 g−1 FW) treated plants, which was 131.58% higher than the inoculated control. The lowest APX activity was recorded in Zn0B1.5 (4.71 µmol min−1 g−1 FW), which was statistically similar to the Z0B3 treatment , which was 30.60% higher than the inoculated control (Z0B0). The AsA content in Zn4B3 + Ar was statistically identical to the treatments of Z4B1.5, Zn4B1.5 + Ar, Zn2B3, Zn0B3, and Zn2B0 in leaves (The AsA levels of sugar beet leaves and roots showed significant variation among the treatments . An incrn leaves A.0B0) in Zn treated plants. Interestingly, a decreased AsA concentration was observed in the roots that were only treated with B .An increased AsA concentration was recorded in the roots of inoculated control compared to the uninoculated control (Znd with B B. The hi−1 FW) in inoculated control (Zn0B0 +Ar) was 35.77% higher than the uninoculated control (0.0123 µmol g−1 FW). Similarly, in roots, a higher MDA concentration (0.137 µmol g−1 FW) was recorded in inoculated control (Zn0B0 + Ar), while a lower amount was found in Zn2B3 (0.0036 µmol g−1 FW) treated plants. However, plants treated with Zn and B significantly decreased the MDA contents both in leaves and roots, even after the inoculation of the pathogen , CAT activity exhibited a decrease compared to the uninoculated control (Zn0B0). It has been suggested that CAT might suppress biotrophic pathogens while encouraging necrotrophic pathogens through host–pathogen interaction [2O2. Other studies have also highlighted the role of CAT in combating various fungal pathogens in important crops such as lettuce, oilseed rape, non-heading Chinese cabbage, and sunflower [We observed an elevation in CAT, APX, and POX activity in Zn treated plants compared to both uninoculated and inoculated plants. CAT activity was notably higher in Zn-treated plants both before and after inoculation of eraction ,52. The unflower ,53,54,55A. rolfsii stress could be attributed to the increased CAT, POD, APX, and AsA activity, which mitigates the harmful effects of ROS generated during pathogen infection. These results are consistent with other studies that Zn amendments or supplementation enhance the activities of various defense enzymes in plants, triggering systemic resistance against pathogens [A. rolfsii.The POX is a well-known pathogenesis-associated protein that helps cell wall lignification and suberization, creating a physical barrier against invading pathogens for localized control ,39. Treaathogens ,20,25,27athogens . ConsequA. rolfsii only slightly enhanced the activity of CAT, POX, and APX in plants grown in B-amended soil, in comparison to both uninoculated and inoculated control plants. The results of the study suggest that B aids in maintaining ROS homeostasis under conditions of B-deficiency by regulating the antioxidant enzyme activity. However, it does not seem to suppress the growth of A. rolfsii through the enhancement of antioxidant defense mechanisms. Boron is an integral part of cell wall structure, and B-deficiency leads to a decrease in the mechanical strength of the cell wall by altering cellulose, pectin, and lignin biosynthesis [Boron is another crucial micronutrient for plant growth and development. Interestingly, an increase in CAT, POX, and APX activity was observed in plants grown in boron-deficient soil (uninoculated control plants) compared to soil amended with B. This observation aligns with findings by Song et al.  and Wangynthesis . Severalynthesis ,25. It iynthesis ,41. Furtynthesis . Hence, A. rolfsii pathosystem through the application of Zn and B. It is evident that both Zn and B help to alleviate the adverse effects of A. rolfsii in sugar beet plants and increase chlorophyll content. Both Zn and B play distinct roles in modifying the antioxidant defense response within sugar beet plants when faced with A. rolfsii infection. Zn has shown the ability to enhance the activity of antioxidant enzymes, whereas B appeared to have less pronounced effects on modulating the antioxidant system to mitigate the detrimental impacts triggered by A. rolfsii. However, a more comprehensive study is needed to explore the antifungal effects of Zn and B on A. rolfsii, examine the regulation of other antioxidant enzymes, and investigate the modulation of beneficial microorganisms in soil by the application of Zn and B in sugar beet cultivation system. This, in turn, could help to mitigate the detrimental impacts of A. rolfsii on sugar beet plants.This study is the first to describe the regulation of ROS in the sugar beet-


Originating in Africa, African swine fever (ASF) was introduced to China in 2018. This acute and highly virulent infectious disease affects domestic pigs. The World Organization for Animal Health has listed it as a statutory reportable disease, and China has listed it as a category A infectious disease.B646L, EP402R, MGF505-3R, and A137R). The primers/probes were highly conserved compared with the gene sequences of 21 ASFV strains.Primers and probes were designed for four ASFV genes (2 > 0.99), the minimum concentration of positive plasmids that could be detected was 50 copies/µL, and the minimum viral load detection limit was 102 HAD50/mL. Furthermore, quadruple quantitative polymerase chain reaction (qPCR) with nucleic acids from three porcine-derived DNA viruses and cDNAs from eight RNA viruses did not show amplification curves, indicating that the method was specific. In addition, 1 × 106, 1 × 105, and 1 × 104 copies/µL of mixed plasmids were used for the quadruple qPCR; the coefficient of variation for triplicate determination between groups was < 2%, indicating the method was reproducible.After optimization, the calibration curve showed good linearity (REP402R, MGF505-3R, and A137R strains with different combinations of gene deletions were as expected. Therefore, the established quadruple qPCR method was validated for the molecular diagnosis of ASF using gene-deleted ASFV strains.The results obtained by testing clinical samples containing detectable The online version contains supplementary material available at 10.1186/s12985-023-02111-1. Asfarviridae family, which has 24 known genotypes [African swine fever (ASF) is a highly contagious viral disease caused by the African swine fever virus (ASFV), a double-stranded DNA virus belonging to the enotypes , 2. Its enotypes . The virB646L gene encoding p72 [EP402R gene encodes a late expressed CD2v protein, a glycoprotein similar to the surface adhesion receptor CD2v on T lymphocytes [EP402R, MGF, and A137R are usually targeted; therefore, establishing corresponding detection methods is necessary for clinical applications.The p72 protein encoded by the B646L gene of ASFV is a major coat protein expressed at a late stage with a differential sequence in the C-terminal region . Thus fading p72 , 2. The ding p72  and I (rphocytes . Multigephocytes . MGF prophocytes  and are phocytes . The A13phocytes . In the The diagnosis of ASFV involves a virus isolation–erythrocyte adsorption assay (HAD), polymerase chain reaction (PCR), real-time fluorescent quantitative PCR (qPCR), and isothermal amplification techniques. Virus isolation is a confirmatory method, and its corresponding assay (the erythrocyte adsorption assay) is time-consuming and can be used only to validate strains with erythrocyte adsorption characteristics. Moreover, it must be conducted in a biosafety level III laboratory to measure viral activity in samples and is dependent on the presence of actively replicating virus, which may be absent if the sample has not been correctly stored, resulting in inactivation, thus limiting its clinical applications . The isoB646L, EP402R, MGF505-3R, and A137R) and established a quadruple fluorescent qPCR assay to diagnose ASF and differentiate gene-deleted strains from wild-type strains.Because using multiple methods and experiments to detect multiple genes is time-consuming and laborious, only a few genes have been detected using the currently available qPCR methods. In this study, we designed primers/probes for four ASFV genes  and were subjected to BLAST analysis. The primers/probes were compared with several ASFV strains published in GenBank using SnapGene software (www.snapgene.com/). The primers/probes . The four recombinant plasmids were mixed so that the concentration of each recombinant plasmid was 2.5 × 108 copies/µL. Next, the mixed plasmids were diluted to 1 × 108 copies/µL, and 10-fold dilutions were performed to obtain 1 × 100 copies/µL; 1 × 102 copies/µL of the mixed plasmids was twice diluted in half to obtain 25 copies/µL. Lastly, clinical and diluted samples from the 108–100 HAD50 ASFV blood series were subjected to nucleic acid extraction using the QIAamp DNA Mini Kit .With reference to the ASFV HuB20 strain (GenBank sequence number: MW521382), full-length Using a LightCycler 480II fluorescent qPCR instrument , 2× HyperProbe Mixture  was selected as the premix required for the reaction, and the primer/probe concentration, annealing temperature, and cycle number were optimized. Next, the total system (Table 6 copies/µL of the mixed plasmid to 102 copies/µL was used as a template for quadruple qPCR amplification. Based on the cycle threshold (Ct) and copy number of the template, a standard curve was generated, and its slope and coefficient of determination (R2) were determined.A 10-fold serial dilution of 1 × 10Sensitivity.3, 1 × 102, 50, 25, and 1 copies/µL were used as reaction templates to test the sensitivity of the quadruple qPCR. In brief, blood samples with a viral load of 108 HAD50 ASFV were diluted 10-fold to 100 HAD50, and nucleic acids were extracted to test the sensitivity of quadruple qPCR for detecting nucleic acid templates representing different viral loads.Mixed plasmids of 1 × 106 to 1 × 104 copies/µL of mixed plasmids were used as templates for triplicate determinations, performed within and between groups of mixed plasmids of each gradient, to test the reproducibility of the quadruple qPCR. The standard deviation and coefficient of variation were calculated.The specificity of the quadruple qPCR was determined using nucleic acids of foot and mouth disease virus (FMDV), bovine viral diarrhea virus (BVDV), porcine epidemic diarrhea virus (PEDV), pseudorabies virus (PRV), porcine parvovirus (PPV), porcine reproductive and respiratory syndrome virus (PRRSV), swine influenza virus (SIV), porcine circovirus II (PCVII), Japanese encephalitis virus (JEV), classical swine fever virus (CSFV), transmissible gastroenteritis virus (TGEV), and ASFV kept in the WOAH reference laboratory of the China Veterinary Drug Inspection Institute. In addition, 1 × 102O, and 2× HyperProbe Mixture is presented in Table The clinical samples collected included blood, liver, spleen, Hubei/2019 genotype II ASFV lung, Genotype 1 ASFV cell samples, and Artificial construction ASFV ΔA137RΔEP402R cell sample. Nucleic acids were extracted from these six samples (200 µL each) and were eluted with 50 µL of eluent, of which 3 µL each was used for the quadruple qPCR. The total system, with each primer/probe, ddHAll treatments for viruses were performed in a Biosafety Level III Laboratory of the China Veterinary Drug Inspection Institute.B646L, EP402R, and A137R of ASFV genotypes I and II. In addition, the MGF505-3R primers/probes were conserved in genotype I Benin 97/1 (AM712239), genotype I OURT 88/3 (NC_044957), HeN/ZZ-P1/2021 (MZ945536), and SD/DY-I/2021 (MZ945537), and in genotype II strain L60 (KM262844)  were 0.9962, 0.9970, 0.9940, and 0.9922, respectively  and RNA viruses  did not show amplification curves in the quadruple qPCR; only the positive control for ASFV showed typical amplification Fig. , indicat6 to 1 × 104 copies/µL of mixed plasmids showed good reproducibility in the three replicates between groups within the quadruple qPCR, with coefficients of variation of less than 2% in all cases , L60 (KM262844), and genotype II ASFV. However, deletions were found in OURT 88/3 (NC_044957) and in the frequently isolated Chinese genotypes HeN/ZZ-P1/2021 (MZ945536) and SD/DY-I/2021 (MZ945537). With this method, we inferred that a sample with positive results for B646L, EP402R, and A137R and negative results for MGF505-3R could contain genotype I ASFV. Moreover, we verified this result with a known genotype I ASFV cytotoxic sample  and eight porcine-derived RNA viruses . None of these 11 nucleic acids showed amplification curves; only the positive ASFV control showed typical amplification see Fig. , indicat2 HAD50/mL. Moreover, our method was more sensitive for the detection of B646L and EP402R than other qPCR methods [Regarding sensitivity, the minimum limit of detection for all four genes was 50 copies/µL and 10 methods . BecauseA limitation of our study is that only four genes could be detected as only a maximum of four fluorescence channels are available in the current fluorescence PCR instruments. In the future these instruments may improve to include more fluorescence channels, which would allow for detection of additional genes.B646L, EP402R, A137R, and MGF505-3R to distinguish ASFV wild-type strains from gene-deleted strains based on current research. This method is the only qPCR method that can simultaneously detect four ASFV genes with conserved primer/probe sequences with high sensitivity, specificity, and reproducibility, providing a comprehensive diagnosis of ASFV.In conclusion, we established a quadruple qPCR method for Below is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2Supplementary Material 3


Nanoscale electro-mechanical systems (NEMS) displaying piezoresistance offer unique measurement opportunities at the sub-cellular level, in detectors and sensors, and in emerging generations of integrated electronic devices. Here, we show a single-molecule NEMS piezoresistor that operates utilising constitutional and conformational isomerisation of individual diaryl-bullvalene molecules and can be switched at 850 Hz. Observations are made using scanning tunnelling microscopy break junction (STMBJ) techniques to characterise piezoresistance, combined with blinking (current-time) experiments that follow single-molecule reactions in real time. A kinetic Monte Carlo methodology (KMC) is developed to simulate isomerisation on the experimental timescale, parameterised using density-functional theory (DFT) combined with non-equilibrium Green’s function (NEGF) calculations. Results indicate that piezoresistance is controlled by both constitutional and conformational isomerisation, occurring at rates that are either fast (equilibrium) or slow (non-equilibrium) compared to the experimental timescale. Two different types of STMBJ traces are observed, one typical of traditional experiments that are interpreted in terms of intramolecular isomerisation occurring on stable tipped-shaped metal-contact junctions, and another attributed to arise from junction‒interface restructuring induced by bullvalene isomerisation. The quest for miniaturisation of electromechanical nanosystems requires the use of single molecules as active components. Here, the authors develop a piezoresistor based on a single bullvalene molecule that changes its shape by a Cope rearrangement. Electrocatalysis can be used to drive reactions6, as the electric field inside the STM can modify molecules7 as well as to measure their properties16, and electric-field driven redox and interfacial reactions have been shown to lead to unprecedented rectification in molecular devices18. In alternate technology, STM junctions have been modelled19 and shown22 to lead to interfacial structural changes through mechanical control of the junction separation. This is of practical significance as it can lead to piezoresistance in a nano electro-mechanical system (NEMS) measured on the Å atomic scale.It is now possible to observe single-molecule chemical reactions using scanning tunnelling microscopy (STM) beyond simple electron-transfer reactions35. Herein, constitutional and conformational isomerism is combined and used to: (i) create fast-switching transistor-like circuits that can exploit single-molecule isomerisation on the ms timescale, (ii) detect oscillating reactions in junctions and use the resulting alternating-current profiles as indicators of junction properties, and (iii) reveal processes leading to junction formation that are critical36 to basic understanding of molecular junctions.Indeed, length changes can be detected by monitoring changes in the junction conductance associated with molecular stretching or junction rearrangement38 molecules  at a rate of 3.2 kHz, to make the structure fluxional. This constitutional isomerism, as well as configurational isomerism associated with the diaryl bullvalene linkages, could control conductance. To investigate this, electrical circuits are formed via the STM break junction (STMBJ) technique in which an Au STM tip is crashed into an Au(111) substrate and then retracted41, allowing single molecules to bridge the broken contacts  approachnce Fig. . This al44. In addition, DFT/NEGF molecular dynamics (MD) simulations of junction extension are performed. These simulations explore possible connections between piezoresistance and bullvalene isomerism, including: (i) constitutional isomerism, which changes through-bond conduction pathways and could also modulate quantum interference effects46, (ii) bullvalene-linkage conformational isomerism, which could also modulate conductance48 and related properties49, (iii) π-stacking of the aryl groups controlled by both constitutional and conformational isomerism that can modulate conductance32 and induce interference effects28, and (iv) junction interface rearrangements24.To interpret these experiments, a kinetic Monte Carlo (KMC) scheme is introduced for the modelling of isomeric composition on the experimental timescale, parameterised by density-functional theory (DFT) coupled with non-equilibrium Green’s function (NEGF) conductance calculations51 for gold‒molecule‒gold junctions. These results are interpreted in terms of isomerisation-driven tip reconstruction bullvalene was prepared from bis(Bpin)bullvalene through a Suzuki cross-coupling reaction38 with 4-bromothioanisole , , and . A network diagram of the ensemble is shown  as isomers A:B:C in the ratio 53:33:13. Results from DFT calculations are consistent with this observation , see Methods. Some current decays, depicting conductance in units of the “quantum of conductance” G0 = Single-molecule conductance was measured using the STM in which an Au STM tip is pushed in and out at a rate of 0.5 Å ms48 is the ratio of the conductances at the high and low half maxima 2)n-SH observed48 under similar experimental conditions. It reflects the typical spread of molecular conductances observed in STMBJ experiments and is traditionally attributed to variability on the gold‒sulfur junction geometry. Much smaller values48 of 53 experiments in which the conductance is measured on ordered self-assembled monolayers (SAMs) with regular interface geometries.A statistical analysis of probability versus the logarithm of the conductance is shown in Fig. para-terphenyl instead of the bullvalene comprising the same S‒CH3 molecule‒electrode contacts are provided in Supplementary Fig. η = 1.1 (blue arrow) and 1.2 (black arrow). These values are less than that typically found for ordered molecular junctions in blinking experiments48 and indicate that the interface geometries that facilitate conduction at low to moderate bias voltages are very regularly reproduced. Figure Uncharacteristic of STMBJ molecular conductance measurements is that a smaller population of the junctions/events form conductance peaks  traces are provided in Fig. −1 with an average duration of 0.7 s for the blinks at the 150 In Fig. para-terphenyl with the same S‒CH3 molecule‒electrode contacts, are provided in Supplementary Fig. Further, the trace at 16.2 Å shown in Fig. A – L in the gas phase, see Supplementary Table A,B, and C are observed in solution NMR, computations are used to investigate their conformational isomerism about the two bullvalene to aryl bonds. Stable conformers with torsional angles near Amm, Amp, Apm, Bmm, Bmp, Bpm, Bpp, Cmm, Cmp, and Cpm, where “p” and “m” indicated positive and negative torsional angles, respectively  timescale to form stable regular tips to which molecules bind. As just one tip profile is commonly used, these models typically do not account for the width . For the low-energy isomers A – C, surfaces were constructed for all four conformers mm,mp,pm, and pp, with the energy of the lowest-energy conformer predicted at each distance only presented in the figure; see Supplementary Table A is predicted to be the dominant isomer, forming tight π-stacked (Amp) and T-shaped (Amm) inter-aryl interactions at short distances down to B and C isomers dominate, joined by E above 19 Å.Critical aspects of the conductance-trace simulations are highlighted in Fig. entclass1pt{minimamm conformers. Most important are the Amm-Dmm, Bmm-Dmm, Bmm-Cmm, and Bmm-Emm transition states as these depict interconversion of the lowest-energy constitutional isomers. From this data, combined with the isomer energetics from Fig. 40 Cope-rearrangement rate in solution of 1.1 kHz per reaction. Note that the rates for forward and reverse reactions will usually differ, and the speedup factors shown are for the slowest of these reactions, with the other process varying from similar rates to rates on the ps timescale. The slowest rates are therefore indicative of the timescale needed to complete oscillating reactions, which from Fig. 33.In Fig. Amm conformation at short tip separations, as a function of tip retraction at the rate used in the experiments of 0.5 Å ms−1. Results are shown in Fig. −1, in good agreement with observed forces for SCH3 – bound gold junctions56 of 0.44 eV Å−1 (0.7 nN). In summary, the dominant constitutional isomers are predicted to change from A at short apex-apex distances to B and C at intermediate distances to E just before dissociation.Using the calculated reaction rates, chemical kinetics equations are then solved, given an initial Next, Fig. Amm conformer at Finally, conductance histograms are simulated and shown in Fig. ned Fig.  and a nened Fig. , in geneAmm isomer . Also, the simulated histogram shows two sharp low-conductance peaks that are atypical of molecular junctions yet identified experimentally in Fig. Bpm contained in the equilibrium isomeric mixtures that exist during the 0.033 ms data-sampling periods.The simulated conductance histogram Fig.  displaysA to D conversion by 0.10–0.13 eV, the third scheme predicts that 70%–90% of conductance traces should dissociate before isomerisation. Nevertheless, the calculations predict that the rate of the A to D conversion should be increased in junctions under tension  no calculated traces indicate dissociation before isomerisation whereas >80% are observed to, and (ii) individual conductance traces show frequent reversible isomerisation, whereas this is rare in the observed traces. Supplementary Note ion Fig.  rather tAmm under tension, which can react to form equilibrium mixtures of Bpm and other isomers at much lower conductance. The 850 Hz reaction is attributed to this equilibrium process, predicted by the calculations to occur at 450 Hz  surface with a 4-atom tip, DFT predicts an activation energy of 0.34 eV for the transfer of the apex atom back one row towards the surface, which, based on the transition-state theory, would be expected to give reaction rates on the 100 ns timescale. 4-atom tips would therefore rearrange before they could be detected by STMBJ, except if the bridging molecule binds faster and inhibits the rearrangement. MD simulations of alkanedithiol junctionsA conformers to compress tightly by utilising strong intramolecular aryl-aryl van der Waals interactions . Shape-shifting bullvalene molecules therefore present many prospects for the development of measurement and control tools for NEMS systems functioning on the Å atomic-length scale.In summary, single diarylbullvalene molecules are observed to undergo constitutional and configurational isomerisation inside an STMBJ, controlled by adjusting the distance between the attached gold contacts. Hence a NEMS piezoresistance device is constructed that is stable on length scales of 3–9 Å that can display factor of 10 changes in conductance. It is anticipated that much larger conductance changes, perhaps exceeding those seen from just pure conformational isomerismA significant property of the bullvalenes is that they can isomerise on the ms timescale to take on vastly different shapes, but this leads to a great many possible atomic-scale data interpretations. The developed DFT/NEGF KMC simulation methodology, and the adapted DFT/NEGF MD methodology, when applied to the prediction of electrode surface structure, molecular binding, molecular isomerisation, and junction conductance, are found to be able to qualitatively interpret all of the major observed features. This provides atomic-level structural and chemical models that can be used to understand function, predict improvements and design new experiments. A basis is therefore provided for the rational improvement of this NEMS technology.As an example of what this technology has to offer for NEMS devices, two characteristically different trace types were found in the STMBJ experiment. This revealed previously inaccessibly information concerning how NEMS interfaces form and vary during experiments. It is reasoned that one type of trace, formed when the bullvalenes stabilise tipped electrode surfaces, is typical of previously assumed STMBJ structures and facilitates isomerisation-driven piezoresestivity. In contrast, the other type of trace is reasoned to be associated with tip flattening induced by bullvalene constitutional isomerism. Tipped surfaces are thermodynamically unstable yet they are believed to dominate the conventional STMBJ structure, and hence the NEMS experiments reveal features of general relevance to molecular electronics applications. In general, the tipped-interface model for STMBJs that have dominated the field for the last two decades needs to be re-evaluated.1H NMR spectra [CDCl3 δ 7.26 ppm] and for 13C NMR spectra [CDCl3 δ 77.16 ppm]. Coupling constants (J) are quoted to the nearest 0.1 Hz. The following abbreviations, or combinations thereof, were used to describe 1H NMR multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, ap. = apparent, br. = broad). IR spectra were recorded neat on a Perkin Elmer spectrum 100 FT-IR Spectrometer with an ATR. ESI-HRMS measurements (electrospray ionisation - ESI) were recorded on an Agilent 6230 time-of-flight LC/MS system. The bis-parathioanisole bullvalene was dissolved in acetonitrile with silver nitrate 0.1% w/v, whereby the [M + Ag]+ value is quoted to Ag107. Melting points were recorded on a DigiMelt melting point apparatus, model MPA161 and were not corrected. Analytical thin-layer chromatography was conducted with commercial aluminium sheets coated with 0.25 mm silica gel . Compounds were either visualized under UV-light at 254 nm, or by dipping the plates in aqueous potassium permanganate or cerium ammonium molybdenite solution followed by heating. Rf values were recorded to the nearest 0.05 cm.NMR spectra were recorded using an Agilent 5600 MHz DD2 console with an Oxford 600 MHz magnet and Agilent cryoprobe, or Bruker UltraShield Avance III 600 MHz NMR Spectrometer. Residual solvent peaks were used as an internal reference for Synthesis of bis(4-thioanisole)bullvalene:2(dba)3  and [HP(t-Bu)3]BF4  in degassed THF (2.5 mL) was added a degassed aqueous 10% NaOH (0.1 mL) solution and stirred for 2 min. 4-bromothioanisole  was then added and the solution stirred for 3 h at 65 °C. The reaction mixture was taken up in water and extracted with DCM (x3). The organics were combined, washed with brine, dried over MgSO4 and concentrated under rotary evaporation. The crude product was purified by silica gel flash column chromatography (0–30% EtOAc/Hex) and then recrystallised from DCM/Hex (1:3) to give the product as a yellow solid .To a solution of bis-Bpin-bullvalene , Pdf: 0.50 (EtOAc/Hex 30:70); m.p. 140–141 °C; 1H NMR  δ 7.33 , 7.29 , 7.21–7.17 , 7.15 , 7.13 , 6.22–6.19 , 6.17 , 6.11–6.01 , 5.95 , 3.53 , 3.19 , 2.84 , 2.79 , 2.62–2.35  ppm;13C NMR : δ = 141.6 (C), 139.8(C), 139.6 (C), 139.3(C), 139.2 (C), 138.3 (C), 136.5 (C), 136.3 (C), 136.3 (C), 128.1 (CH), 127.4 (CH), 126.8 (CH), 126.8 (CH), 126.7 (CH), 126.3 (CH), 126.2 (CH), 125.8 (CH), 125.8 (CH), 125.7 (CH), 124.3 (CH), 123.6 (CH), 122.6 (CH), 39.9 , 35.1 , 29.9 , 23.4 (CH), 21.8 (CH), 21.7 (CH), 20.91 (CH), 20.80 (CH), 15.67 , 15.65 , 15.62 , 15.48 . IR (ATR): v− = 3029, 2922, 1491, 1095, 909, 863, 819, 785, 769, 742 cm−1; HRMS (ESI): m/z calcd. for C24H22S2Ag+: 481.0208 [M + Ag]+, found: 481.0195. NMR spectra are found in the RSynthesis of para - terphenyl):2CO3  in degassed toluene/water (9.8/1.2 mL) was added Pd(PPh3)4 . The solution was stirred and heated at 100 °C for 16 h. The reaction solution was cooled to room temperature, diluted in MeOH, and filtered. The remaining solids were washed with water, then 1 M HCl, followed by MeOH (x4) to afford terphenyl)  as a cream white solidThis compound was synthesised according to a slightly modified procedure taken from ref. To a solution of 4-(methylthio)phenylboronic acid , 1,4-dibromobenzene  and Naf: 0.50 (50% DCM/Hex); 1H NMR  δ 7.64 , 7.57 , 7.35 , 2.54  ppm; 13C NMR  δ 139.5, 137.9, 137.6, 127.5, 127.4, 127.2, 16.1 ppm; MP > 260 °C; HRMS (ESI): m/z calcd. for C20H18S2+: 322.08499 [M]+, found: 322.0844.. NMR spectra are found in the RThe STM measurements were carried out at room temperature in 1,2,4 − trichlorobenzene solvent using 50 µM solution of the diaryl bullvalene with a PicoSPM I microscope head controlled by Picoscan 2500 electronics from Agilent. The STMBJ data were collected using NI-DAQmx/BNC-2110 national instruments (LabVIEW data collection system) and analysed with a home-made code based on the LabVIEW software. Around 3000‒6000 current versus distance curves were collected and accumulated into a conductance histogram. Curves displaying clear plateaus that evidence the formation of a single-molecule bridge were automatically selected and used to build the conductance histograms. An automatic selection driven by a homemade Labview code was used to select the individual traces. The histograms were made by applying the same automated selection criteria to each set of recorded decay curves. The percentage decay curves that showed clear plateaus displaying the occurrence of a single-molecule bridge were typically 40–50% and were all selected to build the histograms. This selection process made peaks in the conductance histograms more prominent above the tunnelling background. Untreated histograms, i.e., without any selection, are presented in the These measurements were made on the same equipment used for the STMBJ experiments. In the blinking approach, the tunnelling current is first stabilised for at least 1 h until a tunnelling current variation of <10% is obtained. Current transients are then captured when a molecule connects between the STM tip and the surface in the presence of a 5 μM solution of the diaryl bullvalene in 1,2,4 − trichlorobenzene. More details of the procedure and its interpretation appear in Supplementary Note 57 using the PBE density functional58 and Goerigk and Grimme’s “D3(BJ)” empirical dispersion correction59; the 6–31 G* basis set60 was used for S, C, and H, with LANL2DZ61 used for Au. Transition states were found by manual grid searching and interpolation utilising the two changing C-C bond lengths. At least every sixth resulting structure was then transferred in between two (4 × 4) 2D periodic models of the Au(111) surface made using an Au-Au bond length of 2.897 Å. large vacuum region was used to separate images perpendicular to the slabs, using a lattice constant of 55 Å. Geometry optimisations were then performed using VASP 5.462, using PAW pseudopotentials63, high precision, and a basis-set energy cutoff of 503 eV. The PBE density functional was used, with D3(BJ) corrections replicated within the 2D plane only. The outside two layers of each slab were frozen during this process, freezing the distance For the calculations using regular 4-atom Au tips, initially optimised structures and transition states were determined using a cluster model. In this, the 2D gold surfaces were ignored, with four frozen atoms in a triplet state used simply to represent the tips. The apex-apex distance was frozen at distance −1. As indicated in Supplementary Figs Calculations for the tip restructuring Figs.  and 8 we44. The molecular electronic junction was divided into three regions, bottom electrode, contact region, and top electrode. The contact region includes parts of the physical electrodes (4 (111) layers of gold), additional electrode atoms to model tip structure, and the bridging bullvalene molecule. All NEGF calculations of molecular junction conductances were performed self-consistently. Used was the PBE density functional, double-zeta with polarisation basis set, k-space grids of 3 × 3 × 1, and 50 a. u. energy cut-off for the real-space grid.The NEGF calculations were performed using Nanodcal64. They solve for the time evolution of the isomeric composition, given the calculated isomeric energies, Cope-reaction transition-state energies, and isomeric conductances, with the tip being constantly retracted throughout the simulations. A small correction was applied to all of the calculated transition-state energies so that the calculated value for bullvalene matched the observed Cope-reaction rate in solution (3.2 kHz net for the 3 possible Cope rearrangements). This approach ignores the change in solvent from CCl2 = CCl2 (mm, mp, pm, and pp conformers of A – E were calculated, as well as that for single conformers of G and K. Conformational equilibrium is assumed throughout the simulations, which focus on the Cope rearrangements only. Transition-state energies were calculated for one conformer only, these being used to approximate the energies for the other conformers. The raw calculated data was interpolated at different values of These were performed by the programme “kinetics_t.for” using input data (from Supplementary Tables Further information on research design is available in the Supplementary InformationPeer Review fileReporting SummarySource Data


Cerebrospinal fluid-based real-time quaking-induced conversion (CSF RT-QuIC) is currently the most prominent method for early detection of sporadic Creutzfeldt–Jakob disease (sCJD), the most common prion disease. CSF RT-QuIC delivers high sensitivity (>90%) and specificity (100%), which has been demonstrated by large ring-trial studies testing probable and definitive sCJD cohorts. Following the inclusion of CSF RT-QuIC in the revised European CJD Surveillance Network diagnostic criteria for sCJD, it has become a standard diagnostic procedure in many prion disease reference or surveillance centers around the world. In this study, we present the implementation of the second-generation CSF RT-QuIC (commonly known as Improved QuIC or IQ) at the Danish Reference Center for Prion Diseases (DRCPD). The method’s sensitivity and specificity were evaluated and validated by analyzing 63 CSF samples. These 63 samples were also analyzed at the National CJD Research and Surveillance Unit (NCJDRSU), based at the University of Edinburgh, UK; analysis was carried out using the first generation or previous CSF RT-QuIC method (PQ). The sensitivity and specificity of PQ during tests at the NCJDRSU were 92% and 100%, respectively. Using these 63 CSF samples, the agreement between the two RT-QuIC generations at DRCPD and NCJDRSU prion laboratories was 100%. Real-time quaking-induced conversion (RT-QuIC) is a method used to detect minute amounts (<10−9 g) of PrPsc in cerebrospinal fluid (CSF) samples, using recombinant Syrian hamster full length PrP  as a substrate. In the presence of pathological PrP, the recombinant PrP substrate undergoes a conformational change, which is detectable by increasing fluorescence emission [Prion diseases are a family of invariably fatal neurodegenerative protein misfolding diseases caused by misfolding of cellular prion proteins (PrPamounts <0−9 g of This CSF RT-QuIC setup is referred to as first-generation RT-QuIC or Previous-QuIC (PQ), in the light of recent development of the second-generation RT-QuIC or Improved-QuIC (IQ).Highly sensitive (>90%) and specific (100%) PQ has been increasingly applied for early diagnostics of sporadic Creutzfeldt–Jakob Disease (sCJD), the most common prion disease ; thus, iRecent ring-trial studies of IQ performance for sCJD diagnostics have demonstrated the method’s high reproducibility and robustness . FurtherIn recent years, RT-QuIC has also been tested for its applicability using different biospecimens , based at the University of Edinburgh, UK. Currently, CSF PQ at NCJDRSU performs with 92% sensitivity and 100% specificity [n = 18, negative sCJD: n = 45) were analyzed in both centers and the concordance of the results was 100%. In this paper, we describe our workflow and provide detailed information on the establishment of this highly sensitive and specific method; that is now used for early sCJD diagnostics also in Denmark. At the DRCPD, we have implemented and validated IQ for sCJD diagnostics. Our IQ validation process included cificity . The samin-house-produced rHaTrPrP, we established criteria for batch purity, stability, specificity, and sensitivity. rHaTrPrP purity is estimated by Coomassie SDS-page analysis, which must indicate a single band at 16.2 kDa and no clearly visible contaminants  decrease in sCJD brain homogenate (BH) controls. We tested if the RFU decrease over time was influenced by BH freshness; this was not the case, as even with a freshly prepared control, the RFU was lower with the older rHaTrPrP. To determine the positivity of each well (replicate), a threshold for a cut-off value was established using 23 out of 45 negative (non-CJD) CSF samples. All 23 samples were analyzed in quadruplicates with both 15 µL and 30 µL CSF. Thus, 184 replicates were used in total to determine the mean RFU value. RFU was measured every 45 min for 50 h (65 data points), and the determined mean was 203 RFU with a standard deviation of 65.35. Therefore, a cut-off value for a positive replicate was set at 900 RFU, which approximately equals the mean RFU + 10 standard deviations.To define the mean RFU for a CJD-positive IQ, we used RFU values from the two best-performing (maximum RFU) replicates from each of the 18 CSF samples (30 µL) from patients with probable sCJD. A distribution pattern of the mean RFU is depicted in Sc in the sample, but it cannot be ruled out that other factors, such as relative salt concentration in CSF compared to that of the IQ mastermix composition, play a role. However, when considering all 72 replicates of the 18 samples with probable CJD, the mean curve for both 15 µL and 30 µL CSF volume still surpasses the set RFU cut-off value  was commercially ligated , with DNA sequences corresponding to the prion protein gene (PRNP) sequence encoding amino acids from 90 to 231 in the Golden Syrian hamster . Ligation was performed to strategically bypass the N-terminal His-tag of the PRSET plasmid. BL(21)E3 chemically competent cells were transformed, and appropriate colonies were selected for glycerol stocks. A tip of glycerol stock was used to inoculate 5 mL LB media at 37 °C, 220 rpm, 5–7 h. The LB media-containing cultures were diluted 1:500 in 4 × 500 mL auto-induction TB media, and incubated at 37 °C, 200 rpm, overnight, in non-baffled flasks. Cells were pelleted at 3200× g, 10 min, RT, and divided into six 50 mL tubes. Cell pellets were washed with 2× BugBuster Mastermix (15 mL/10 g wet cells) using mild homogenization and allowed to gently mix by rotation for 10–30 min, RT. Cells were then pelleted at 13,000× g, and the above-described extraction was repeated 2 times with 10% BugBuster Mastermix. The resulting white pellets were totally solubilized in denaturing buffer  (30 mL/cell pellet) by rotation for 24–72 h, 10 rpm, 4 °C. Ninety mL  of Ni-NTA Superflow  was washed with a denaturing phosphate buffer  by rotation at 10 rpm, 3 × 10 min, followed by centrifugation at 1500× g and discarding of the supernatant. The resulting slurry was equilibrated with the rHaTrPrP solution and allowed to bind by rotation at 10 rpm, 1 h, RT. The slurry was then pelleted and packed in a suitable column onto an ÄKTA FPLC, at 10 mL/min, 10 min. rHaTrPrP was folded with a linear gradient  over 200 min at 5 mL/min flow. The product was eluted with a linear imidazole gradient  over 45 min at 6 mL/min and collected as a broad peak between 250–350 mM imidazole. The elute was sterile filtered (0.2 µm), while the concentration was estimated with spectrophotometer at Abs280 (250 mM imidazole as base line) and subsequently diluted to 0.8 mg/mL with a 10 mM phosphate buffer, pH 6.5. The elute was dialyzed extensively against a 10 mM phosphate buffer at pH 6.5 (>20 times the elute volume), and the final product concentration was estimated by QubitTM 4 fluorometer using protein assay. The batch is then properly aliquoted on ice, flash frozen with liquid nitrogen and stored at −80 °C. The yield of the batch used for this study was ~70 mg. The typical range over four batches was 40–70 mg. rHaTrPrP was produced idelines . More spTM imaging platform with stain-free settings. To estimate the purity of produced rHaTrPrP, samples with a total volume of 20 µL  were heated at 85 °C for 5 min and loaded on a 10-well 10%, Bis-Tris, 1.0 mm gel. The gel was then stained with 200 mL Coomassie stain  for 15 min, 300 rpm, in microwaved (1 min) stain solution. De-staining  was repeated twice for 15 min, 300 rpm, in microwaved (1 min) 200 mL de-stain solution. Stained gel was visualized on ChemiDoc−7. The threshold for deviation from expected results was set at <5%, i.e., 1/32 reactions could yield a false positive or negative result. The sensitivity of the rHaTrPrP produced following the protocol described in w/v sCJD BH ranging from 10−5–10−15. This was only performed for the initial batch and is not included in our batch-to-batch evaluation.The stability of newly produced rHaTrPrP was evaluated by plate setup consisting of 32 wells of unseeded mastermix, Alzheimer’s Disease (AD) BH and sCJD BH at a dilution of 10TM (BMG® LABTECH) plate reader with continuous cycle of 60 s. shaking, at 700 rpm, and 60 s. resting, for total of ~48 h, at 55 °C. A fluorescence measurement, expressed in RFU at 450 ± 10 nm excitation and 480 ± 10 nm emission, was taken every 45 min. Gain was set to 1000. Each black, 96-well plate with a flat and transparent bottom, was loaded with unseeded mastermix, sCJD BH, AD BH, a non-CJD and probable CJD CSF samples (15 µL and 30 µL). All controls and samples were analyzed in quadruplicates. The plate was then sealed with optical adhesive film and incubated in an Omega FLUOstarIQ results for a quadruplicate setup were interpreted as either negative 0/4), inconclusive (1/4) and repeated, or positive (2–4/4) [, inconclPRNP sequencing, tissue morphology and immuno-histochemistry. Frozen brain samples with a post mortem-confirmed diagnosis of sporadic CJD MM1 and AD were used for the preparation of a positive and negative BH control, respectively. AD cases were confirmed to be negative for prions by immuno-histochemistry. BH controls were prepared as a 10% w/v in TBS-based extraction buffer and homogenized using DispomixTM tissue homogenizer for 15 s, 4000 rpm. The homogenates were then aliquoted to 100 µL and stored at −20 °C. The freeze–thaw cycles of each control were recorded; these cycles did not occur more than five times. BH were serially diluted to 10−15 in sterile filtered (0.2 µm) 0.1% SDS in PBS, at pH 7.4, to determine the best performing concentration, which subsequently was used as a control for diagnostic IQ.At DRCPD, samples collected from certain brain areas, i.e., frontal cortex (FC) and cerebellum (CB), were immediately preserved by freezing them at −80 °C. The rest of the brain was formalin-fixed  for a month, treated with formic acid for 1 h and paraffin-embedded. The definitive diagnosis was provided based on prion detection by immunoblotting, ante mortem) were included in this IQ validation. Overall, 48 of these samples were delivered to DRCPD for diagnostic purposes from several neurological departments in Denmark. Samples were shipped on dry ice and, upon arrival, stored at −80 °C. The remaining 15 samples were from patients with probable sCJD and provided by NCJDRSU [n = 14 at 30 µL, n = 1 at 15 µL) and RFU for the two best-performing replicates for each sample was provided.In total, 63 CSF samples  were composed of a 98 µL mastermix  and 2 µL serially diluted pre-vortexed BH (10−7). For unseeded controls, 2 µL 0.1% SDS in PBS at pH 7.4 were added instead of BH. CSF samples  were composed of a 70 µL or 85 µL mastermix , and 15 µL or 30 µL neat pre-vortexed CSF, respectively. All Mastermix buffers and solutions were freshly prepared on the day of each RT-QuIC assay, sterile filtered (0.2 µm) and kept cool until use. rHaTrPrP aliquots were thawed at RT and moved to 4 °C. The recombinant protein was spin filtered through a 100 kDa MWCO at 4000× TM Omega Plate Reader (BMG® LABTECH) was connected to Omega Control and Omega MARSTM software for instrument control and data analysis, respectively. Using Omega MARSTM, multiple plates were merged into one layout, and all data were exported as a table sheet of all cycles for statistical data analysis in GraphPad® PrismTM 9.FLUOstarµ) of all replicates and standard deviation (σ) for all measured cycles, except 0 min, was calculated. The threshold for a positive CSF was set at the RFU corresponding to +10 standard deviations. Confidence Interval (95%) was based on the standard error of the mean of all replicates.A cohort of non-CJD CSF samples was used to establish a RFU cut-off value for a positive sample. A mean (


Culex quinquefasciatus mosquitoes  and three urine samples collected from patients in three different provinces of Thailand. Additionally, successful isolation of a ZIKV isolate occurred from a female Cx. quinquefasciatus. The WGS analysis revealed a correlation between the 2020 outbreak and the acquisition of five amino acid changes in the Asian lineage ZIKV strains from Thailand (2006), Cambodia (2010 and 2019), and the Philippines (2012). These changes, including C-T106A, prM-V1A, E-V473M, NS1-A188V, and NS5-M872V, were identified in all seven WGS, previously linked to significantly higher mortality rates. Furthermore, phylogenetic analysis indicated that the seven ZIKV sequences belonged to the Asian lineage. Notably, the genomic region of the E gene showed the highest nucleotide diversity (0.7–1.3%). This data holds significance in informing the development of molecular tools that enhance our understanding of virus patterns and evolution. Moreover, it may identify targets for improved methods to prevent and control future ZIKV outbreaks.Zika virus (ZIKV), a mosquito-borne flavivirus, has been continually emerging and re-emerging since 2010, with sporadic cases reported annually in Thailand, peaking at over 1000 confirmed positive cases in 2016. Leveraging high-throughput sequencing technologies, specifically whole genome sequencing (WGS), has facilitated rapid pathogen genome sequencing. In this study, we used multiplex amplicon sequencing on the Illumina Miseq instrument to describe ZIKV WGS. Six ZIKV WGS were derived from three samples of field-caught  The ZIKV genome spans approximately 10.8 kb in length and comprises a positive-sense, single-stranded RNA molecule2. The genome organization of flaviviruses follows the sequence 5′-C-prM-E-NS1-NS2a-NS2b-NS3-NS4a-2K-NS4b-NS5-3′4. The structural components of the genome include the capsid protein (C), precursor membrane protein (Pr/M), and envelope protein (E). In addition, seven non-structural proteins (NS) play a critical role in genome replication5.Zika virus (ZIKV) is an emerging RNA flavivirus transmitted by mosquitoes and is closely related to yellow fever virus (YFV), Japanese encephalitis (JEV), dengue virus (DENV), and West Nile virus (WNV)7. ZIKV is mainly transmitted through mosquito vectors, predominantly Aedes mosquitoes8. However, ZIKV can also be transmitted by other routes9, including perinatal vertical transmission from mother to fetus10, blood transfusion11, and sexual transmission12. Sporadic cases of ZIKV have been reported annually in Thailand since 2010, mainly during the annual rainy season13. The rapid spread of ZIKV to 51 countries, including Thailand14, occurred between 2016 and 2017, with the Thai Ministry of Public Health recording over 1698 confirmed cases of ZIKV infection (1121 cases in 2016 and 577 cases in 2017)13. Previous studies in Thailand have identified ZIKV RNA in mosquitoes, including Aedes aegypti, Culex quinquefasciatus, and Armigeres subalbatus, collected from the homes of actively infected ZIKV patients16. In 2018, WGS was conducted on two ZIKV isolates from the blood of two Thai patients, showing genetic similarities to isolates from ZIKV outbreaks in Brazil during 2015–201617. A subsequent report revealed that ZIKV has been circulating in Thailand since around 2002 and has significantly spread18. This conclusion was determined from an analysis of the spatial and age-related distribution of cases, along with the construction of time-resolved phylogenetic trees using genetic data from Thailand and other areas18. Following a decline in the number of ZIKV cases from 273 cases in 2019 to 239 cases in 2020, and 63 cases in 2021, there has been a recent increase in cases, reaching 190 cases in 2022 and 479 cases in 202313. This resurgence highlights the urgency of studying the severity of diseases caused by ZIKV. The virus’s rapid spread necessitates an immediate and comprehensive understanding to address the emerging public health concerns.Recently, ZIKV has experienced rapid global spread and has been associated with human diseases such as microcephaly and other birth defects in neonates, as well as Guillain–Barré syndrome in adults19.The recent emergence of high-throughput sequencing technologies, notably whole genome sequencing (WGS), now enables the rapidly acquisition of complete pathogen genomes20. Phylogenetic analyses play a crucial role in elucidating the evolutionary dynamics and dissemination patterns of ZIKV, with two major lineages recognized as the African and Asian lineages based on their initial discoveries in the respective geographic regions21. Examining genetic diversity among ZIKV lineages and across the genome offers valuable insights into the current genotype of the circulating virus22. Several investigations have reported nucleotide (nt) mutations and amino acid (aa) changes throughout the entire diversity of ZIKV25. The presence of specific mutations in all sequences of a specific clade in the phylogeny may hold significance for the control of disease, especially if that clade is associated with increased pathogenicity. However, it is crucial to validate the causal relationship between a mutation and a phenotype through reverse genetics.For ZIKV diagnosis, the recommended gold standard is molecular-based detection of ZIKV RNA in samples, employing methods such as reverse transcription PCR (RT-PCR), real time RT-PCR and nested RT-PCR assay across several alternative specimen typesTherefore, our objective was to characterize ZIKV genetic diversity and perform phylogenetic analyses of positive ZIKV samples through complete genome sequence using multiplex amplicon sequencing-based next-generation sequencing (NGS) on an Illumina sequencing system (MiSeq). Scaling ZIKV whole-genome sequencing presents a valuable opportunity to understand the genetic diversity, evolutionary dynamics, and transmission patterns of ZIKV. The insights gained from this study can be applied to further develop of new molecular tools, and the nucleotide sequence data can contribute to the future development of more effective, region-specific prevention, diagnostics, and control strategies for ZIKV in Thailand.Cx. quinquefasciatus (n = 326), Ae. aegypti (n = 154), Ae. albopictus (n = 10), and Ar. subalbatus (n = 39), were collected from the residences of active ZIKV patients in Bangkok, Chanthaburi, and Nakhon Ratchasima provinces. Hemi-nested RT-PCR (hn-RT-PCR) was employed to screen the presence of ZIKV in both mosquitoes and urine samples from patients with active ZIKV infection. ZIKV was detected in 29 out of 529 samples (5.5%), distributed across Cx. quinquefasciatus , Ae. aegypti  and Ar. subalbatus  , cytopathic effects (CPE) can be observed in one sample of ZIKV isolates derived from mosquitoes  in length. The ZIKV genome encodes three structural proteins: C 365 nt), Pr/M (502 nt), E (1511 nt), and seven non-structural proteins: NS1 (1055 nt), NS2A (677 nt), NS2B (389 nt), NS3 (1850 nt), NS4A (380 nt), 2 K (68 nt), NS4B (752 nt), and NS5 (2708 nt). The complete genome sequences of ZIKV have been deposited in GenBank under the accession numbers OR535163-OR535169. Sequence analysis of ZIKV revealed that our seven genome sequences were related to ZIKV sequences isolated from the serum of patients in Thailand during the outbreak of ZIKV in 2006–2017, Cambodia in 2010 and 2019 (accession no. MH158236.1 and ON209935.1), and the Philippines in 2012 (accession no. KU681082.1) , showcasing 12 amino acid mutations when compared to Cambodia in 2010 (CAM/2010). These mutations include C‐T106A, prM‐V123A, prM‐N130S, prM‐M151L, E‐V763M, NS1‐A982V, NS2A‐A1204T, NS2A‐P1274L, NS2B‐A1477T, NS5‐V2878A, NS5‐M3392V, and NS5‐V3403M. Notably, some of these mutations were associated with neurovirulence42. Furthermore, the CAM/2019 virus showed an NS2A-A1204T mutation closely related to the Thai isolate SI-BKK02 (accession no. MF996804.1), collected from Thailand in 2017 and linked to a case of microcephaly43. Amino acid changes reported in Asian-lineage ZIKV strains from 2010 to 2013 encompassed C-T106A, prM-V1A, prM-S17N, E-V473M, NS1-A188V, NS5-M114V, and NS5-M872V47. Yu et al.48 discovered that the C-T106A mutation heightened infectivity and accelerated the spread of ZIKV in both mosquitoes and rodents, enhancing transmissibility between vectors and hosts. The NS1-A188V mutation exhibited a modest impact on ZIKV infectivity in laboratory Ae. aegypti mosquitoes. Additionally, this mutation was found to inhibit the production of type I interferon (IFN) in mammals45. According to Shan et al.49, mutants C-T106A, E-V473M, and NS5-M872V associated with significantly elevated mortality rates. In particular, the E-V473M mutation was identified to amplify ZIKV replication, resulting in enhanced neurovirulence, increased transmission from mother to fetus during pregnancy, and the presence of viremia in both mouse and non-human primate models49.Our study demonstrated five mutations in all seven ZIKV genome, including C-T106A, prM-V1A, E-V473M, NS1-A188V, and NS5-M872V. Zhang et al.51. These considerations elucidate why our study exclusively focused on urine samples.In this study, we conducted a comparative analysis of the genetic diversity offered by ZIKV mutants containing distinct stable substitutions that emerged within the Asian ZIKV lineage. Genetic variation can provide crucial insights into ZIKV biology and pathogenesis, potentially uncovering functional mutations in the virus. However, the potential relevance of these mutations in this study remains uncertain. The study is limited by a relatively small sample size, encompassing only seven complete genomes collected from different geographical regions in Thailand. The sample collection methodology relied on a report from the Department of Disease Control, Ministry of Public Health, which tested blood samples for ZIKV RNA presence. In general, we collected the mosquitoes in and around the residences of active ZIKV patients whose blood tested positive for ZIKV RNA. Consequently, upon field arrival, we could collect only non-invasive samples such as urine from patients in each area. Moreover, research suggests that ZIKV detection in urine is more sensitive and has a longer window of detection compared to serum and salivaTo comprehensively assess the transmissibility of mutant ZIKV strains within mosquitoes, further investigation using a mosquito-mouse transmission model is necessary. This study not only establishes a scientific basis for understanding the evolution of the ZIKV in Southeast Asia but also illustrates the importance of continued monitoring in future outbreaks of ZIKV. Emphasizing diagnostic challenges, surveillance, and implementing effective control and prevention strategies for ZIKV becomes paramount in stopping the rapid spread of the epidemic in the future.Adult mosquitoes were collected both inside and outside the residences of ZIKV-infected patients during the 2020 ZIKV outbreaks in Bangkok, Chanthaburi, and Nakhon Ratchasima using a backpack aspirator . All mosquitoes were classified according to their sex and species using morphological identification. Urine samples from ZIKV-infected patients were collected. Subsequent testing for ZIKV infection was conducted on both individual mosquitoes and human samples.g for 10 min. Then, 200 µl of the supernatant was transferred to a sterile 1.5 ml microcentrifuge tube mixed with 200 µl of 2X minimum essential medium Eagle (MEM)  containing 2% heat-inactivated FBS , 2% penicillin (100 U/ml), and streptomycin (100 µg/ml) , and stored at − 80 °C for further virus isolation attempts. The mosquito carcass or human urine sample was mixed with 300 µl of lysis buffer and processed for viral RNA extraction using an Invisorb Spin Virus RNA Mini Kit . The mosquito or human urine RNA samples were amplified for detection of ZIKV at NS5 gene using hn-RT-PCR 15. The positive control was constructed and used the lower band of a synthetic positive control plasmid. The amplified PCR products were separated on a 1.5% agarose gel, stained with ethidium bromide, and visualized using Quantity One Quantification Analysis Software Version 4.5.2, Gel Doc EQ System .Individual mosquitoes were placed in 1.5 ml microcentrifuge tubes with 200 µl of 1X phosphate buffered saline (PBS), ground, and centrifuged at 13,000×Ae. albopictus C6/36 cells in 12-well plates, then gently mixed at room temperature for 1 h. The supernatant was removed and replaced with 2 ml of MEM containing 1% heat-inactivated FBS , penicillin (100 U/ml), and streptomycin (100 µg/ml) . The plates were incubated at 28 °C and 5% CO2. CPE was monitored daily for 6–7 days. The hn-RT-PCR amplification of CPE-positive culture supernatant on day 7 confirmed ZIKV infection. The positive isolates were propagated in C6/36 cells cultured in MEM with 10% heat-inactivated FBS  at 28 °C and 5% CO2 for three more passages. Harvested supernatant was kept at − 80 °C until use.The supernatant from ZIKV-PCR positive samples was passed through 0.2 µm syringe filters and seeded onto monolayers of At 0, 3, 5, and 7 days post-infection (dpi), infected cells grown on glass coverslips were harvested and rinsed with 1X PBS (3 × 5 min), fixed with 4% formaldehyde for 15 min at room temperature, and then incubated for 15 min in 0.1% of Triton X-100 in 1X PBS for 15 min at room temperature. After washing with 1X PBS (3 × 5 min), the fixed cells were incubated with a 1:500 primary rabbit-Zika virus NS1 protein antibody , rinsed with 1X PBS (3 × 5 min), and incubated with 1:10,000 Goat Anti-Rabbit IgG H&L (Alexa Fluor 488)  at room temperature for 1 h as the secondary antibody and then washed with 1X PBS (3 × 5 min). Finally, a drop of Prolong gold antifade  was added on each slide and topped with a cover slide. The non-infected C6/36 cells were used as a negative control in the experiment. All slides were examined under a fluorescence microscope .20. The PCR products from 2 primer pools were purified and end-repaired using KAPA Hyper prep kit . The index adaptor was then added to the end of the fragments and PCR was performed. The DNA library was checked for quality and quantity using QIAxcel screen gel  and Qubit high sensitivity DNA kit , respectively. The concentration of each DNA library was calculated. The pooled DNA library was then loaded into the MiSeq reagent kit V3  and sequenced using the MiSeq machine .RNA from positive samples were synthesized for cDNA using Superscript IV enzyme  followed by ZIKV multiplex PCR of 2 primer pools, long‐amplicon 35‐plex PCR primer panel with ~ 11,000‐bp amplicon lengths for full‐genome sequencing of ZIKVhttps://mafft.cbrc.jp/alignment/server/)52. Based on the complete genome sequence of ZIKV from this study and reference strains obtained from the GenBank database. The phylogenetic trees were constructed using the neighbor-joining (NJ) method and maximum composite likelihood model, based on the general time reversible (GTR) model in MEGA 11 software53. Bootstrap values were estimated for 1000 replicates. The phylogenetic trees were edited using Interactive Tree Of Life (iTOL) v6.354. Variation and mutation in the genome are confirmed by manually checking the alignment with ZIKV reference genome from Brazil (accession no. NC035889.1) in Unipro UGENE v47.055.The genome sequences were assembled using multiple alignments using fast Fourier transform (MAFFT) version 7 . The Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand (COA No. 025/2564) approved this study, which abided by the Animals for Scientific Purposes Act and all relevant institutional policies and regulations regarding animal care and use at Chulalongkorn University. The use of hazardous agents was only initiated after approval from the institutional animal care and use committee (IACUC), Institutional Biosafety Committee (IBC), and/or Environmental Health and Safety Department. The use of human blood was approved by the Institutional Review Board of the Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand (COE No. 016/2017), and the study was conducted in compliance with the international guidelines for human research protection as stated in the Declaration of Helsinki, The Belmont Report, the Council for International Organizations of Medical Sciences (CIOMS) guidelines and the International Conference on Harmonization in Good Clinical Practice (ICH-GCP). Informed consent was obtained from all participants. All experimental protocols requiring biosafety were approved by Institutional Biosafety Committees (IBC) of the Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand (MDCUIBC002/2017).